Cytological imaging systems and methods

ABSTRACT

The present invention relates to the analysis of specimens. Specifically, the invention relates to methods and apparatus for reviewing specimen slides, including apparatus for holding the slides. The invention also relates to an automatic focusing method for an imaging system and methods for accommodating vibration in the imaging system. In particular, the methods and apparatus may be applied to the automated analysis of cytological specimen slides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/008,379, filed Nov. 5, 2001 now U.S. Pat. No. 7,369,304, which claimsthe benefit of provisional U.S. Patent Application Ser. No. 60/245,971,filed Nov. 3, 2000. This application is also a continuation-in-part ofU.S. patent application Ser. No. 09/430,198, filed Oct. 29, 1999 nowU.S. Pat. No. 7,006,674, and U.S. patent application Ser. No.09/430,116, filed Oct. 29, 1999 now U.S. Pat. No. 6,348,325, thedisclosures of which are hereby incorporated herein by reference intheir entirety.

This application is also related to U.S. patent application Ser. No.11/236,407, filed Sep. 26, 2005 and U.S. patent application Ser. No.11/236,220, filed Sep. 26, 2005. This application is further related toU.S. patent application Ser. No. 11/861,227 and U.S. patent applicationSer. No. 11/861,230, all of which are filed on the same date herewith.The disclosures of the foregoing applications are expressly incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to systems and methods for the analysis ofspecimens. Specifically, the invention relates to a review system forreviewing specimens, methods of focusing the specimens in an imagingsystem, and apparatus for holding the specimen.

BACKGROUND INFORMATION

Cytology is the branch of biology dealing with the study of theformation, structure, and function of cells. As applied in a laboratorysetting, cytologists, cytotechnologists, and other medical professionalsmake medical diagnoses of a patient's condition based on visualexamination of a specimen of the patient's cells. A typical cytologicaltechnique is a “Pap smear” test, in which cells are scraped from awoman's cervix and analyzed in order to detect the presence of abnormalcells, a precursor to the onset of cervical cancer. Cytologicaltechniques are also used to detect abnormal cells and disease in otherparts of the human body.

Cytological techniques are widely employed, because collection of cellsamples for analysis is generally less invasive than traditionalsurgical pathological procedures such as biopsies, whereby a tissuespecimen is excised from the patient using specialized biopsy needleshaving spring loaded translatable stylets, fixed cannulae, and the like.Cell samples may be obtained from the patient by a variety of techniquesincluding, for example, by scraping or swabbing an area, or by using aneedle to aspirate body fluids from the chest cavity, bladder, spinalcanal, or other appropriate area. The cell samples are placed insolution and subsequently collected and transferred to a glass slide forviewing under magnification. Fixative and staining solutions aretypically applied to the cells on the glass slide, often called a cellsmear, for facilitating examination and for preserving the specimen forarchival purposes.

Typically, screening of cytological specimens has been a task fortrained cytotechnologists and cytopathologists. Even though screening isdone by highly trained individuals, the task is repetitive and requiresacute attention at all times. Therefore, screening of cytologicalspecimens is repetitive and tedious and would benefit from automation;however, the complexity and variety of material found in cytologicalspecimens has proven very difficult to reliably examine in an automatedfashion. Various image analysis systems have been developed foranalyzing image data of specimens taken from a patient to augment thephysician diagnosis of the biomedical status of the patient. Forexample, image analysis systems have been developed for obtaining imagedata representing blood cells, bone marrow cells, brain cells, etc.Image analysis systems are typically designed to process image data todetermine characteristics of the specimen. These systems have been usedprimarily as prescreening systems to identify those portions of aspecimen that require further inspection by a human, i.e., re-screening.

Methods and apparatus for re-screening slides are either very crude orentail great economic expense. The prevailing method in aidingrelocation is the placement of an ink dot on the specimen near thelocation of the event. This method can be crude, awkward, timeconsuming, and inaccurate. In addition, with this method, it is notpossible to ascertain if the entire specimen area of the slide has beenuniformly examined or if areas of the specimen have or have not beenscanned. It is, accordingly, often the case that if the user isinterrupted, it is necessary to restart slide examination. Furthermore,with microscope examination of items for identifying characteristics,the use of ink dots can actually detrimentally impair examination of theitem of interest.

In a laboratory, for example a cytology laboratory, a cytotechnologistexamines numerous specimen slides under a microscope in order to analyzecertain specimen cells of questionable nature. When such suspect cellsare located, the cytotechnologist generally marks the slide at thatpoint, so that he or she may recall the location of the cells at somelater time for further examination. To date, cytotechnologists havemarked slides generally by using one of several manual methods.

One such method exists where a cytotechnologist marks the area of themicroscope slide in question with a marking pen. To accomplish this, thecytotechnologist must take his or her eyes away from the eyepiece of themicroscope, move the microscope objective out of the way, peer under thenosepiece, estimate the location of interest on the slide, and then markthe slide with the pen. This method requires the cytotechnologist torefocus her eyes, move her body into a potentially awkward position, andto make a guess as to the placement of the mark. This method of markingcan be time consuming and is typically not exact.

Another such method consists of marking a microscope slide by using anobjective-like configuration marker. In this method, thecytotechnologist must take her eyes away from the microscope eyepiece,rotate the microscope nosepiece until the marking apparatus is in place,and then manually push the marking apparatus down onto the slide inorder to mark the area in question. This method requires thecytotechnologist to refocus her eyes and to move her body into apotentially awkward position. Although this method is more exact thanthe previously described method, it is still tedious and time consuming.

Additionally, many imaging systems that can be used for imaging aspecimen must be built to prevent vibration from affecting the imagingof the specimen. Image blur, due to vibration, results if the imagingsystem is not sufficiently rigid or dampened. A blurred image istypically unusable. Manufacturing a sufficiently rigid dampened systemcan be costly and adds weight and complexity to the imaging system.

Imaging systems also require apparatus for handling and holdingspecimens. Devices for holding specimens, slides, or similar objects ina defined position relative to an optical instrument, such as an imagingsystem, have been in existence for many years. In the instances of slideholding mechanisms, these features have been incorporated into thestages of automated microscopes so that a slide may be moved with thestage relative to the viewing field of the microscope. Many of theseslide holding devices do not facilitate repeatably holding the slide inthe same precise location in the slide holding area. In someenvironments, it may be desirable to view the slide through the imagingsystem on more than one occasion or to view the slide on differentimaging devices. Being able to repeatably position a slide relative to apredefined coordinate system is a useful feature of the slide holderassembly.

In particular, when the platform holding the slide is undergoingrepeated substantially planar motion to allow the imaging of selectedregions of the slide, there needs to be a reliable system for the secureholding and release of a given slide. When large numbers of slides areused in an automated or semi-automated specimen analyzing apparatus, theability to quickly load, secure, and remove slides from an imagingsystem in a precise and controlled way becomes an advantageous featureof large scale batch sample analysis.

In order for an automated system for analyzing a specimen on a sampleslide to be effective, each image obtained should be in proper focus.Conventional focusing apparatus and methods, however, may betime-consuming, thereby making analysis of sample slides inefficient.Due to variations in distance between positions on the sample slide andimaging optics, focus should be adjusted accordingly during automatedimaging of the slide. There is a need for a system that quickly andaccurately focuses and scans substantially all of an area of interest ofa sample slide. Imaging and analysis may accompany or follow such ascan, whereby specific regions of interest of the sample slide areautomatically denoted and subsequently presented to a cytotechnologist,for example, for further analysis.

SUMMARY OF THE INVENTION

Generally, the invention addresses the problems outlined above byapplying automated methods and related apparatus for screeningspecimens. Such methods and apparatus are used for holding thespecimens, reviewing and marking specimens, efficient focusing ofspecimens, and compensating for vibration. The term specimen is usedthroughout the specification to represent the material being imagedand/or reviewed and is not limited to cytological material. Also, theterms specimen sample, and slide may be used interchangeably throughoutthe specification and figures.

In one aspect, the invention relates to an apparatus for marking aspecimen. The apparatus includes an optical instrument, a marker coupledto the optical instrument and disposed outside a field of view of theoptical instrument, and a stage coupled to the optical instrument forreceiving the specimen. The stage moves the specimen between aninspection position in an optical path of the optical instrument and amarking position for marking the specimen with the marker. The opticalinstrument includes a motorized nosepiece, an illumination source, andfocusing optics. The optical instrument can be in data communicationwith a computer. Alternatively, the optical instrument can be alaboratory microscope.

In various embodiments, the motorized nosepiece includes at least onelens and the lens can include a 10× objective. The motorized nosepiececan include a second lens and the second lens can include a 40×objective. The optical instrument can also include a specimenidentification reader, which can be a bar code reader or an opticalcharacter recognition (OCR) device. Also, the marker can be a nib forapplying a marking substance to the specimen. In some embodiments, theapparatus includes a cap mechanism coupled to the optical instrument andengageable with the marker. The cap mechanism is biased away from themarker when disengaged therefrom and actuatable into an engagementposition with the marker by a pin disposed on the slide stage. The capmechanism can include a resilient seal for sealing the nib of themarker. The apparatus can also include an actuator for moving the markerrelative to the stage. The actuator can be a solenoid, a motor, afluidic cylinder, or other suitable device.

In other embodiments, the apparatus can include a user interface controlin electrical communication with the optical instrument and a console inelectrical communication with the optical instrument. The user interfacecontrol can include at least one input device and a stage positioningdevice, such as a joystick. The input device can perform a variety ofoperations, such as advancing the specimen to a next position in a fieldof view of the optical instrument, advancing the specimen to a previousposition in the field of view, toggling between the first lens and thesecond lens, and marking an object of interest. In addition, the userinterface control can include four switches or other input devices, eachperforming one of the aforementioned operations. The console can includea keypad and a display. The apparatus can also include an audio outputdevice in electrical communication with the optical instrument, such asa speaker for emitting an audible warning tone or instructions to auser.

In some embodiments, the apparatus also includes apparatus forelectronically marking an object of interest within the specimen and anindicator for indicating marked status of a field of interest. Theindicator includes an optical path originating at a light source andpassing serially through a diffuser, a mask, an aperture, focusingoptics, a lens, and a beam splitter. All of the elements are disposedwithin a housing. The light source can include two separate lightsources. The optical instrument can further include a sensor fordetecting presence of a specimen. The sensor can be a proximity switch,a limit switch, a hall-effect switch, a magnetic sensor, an opticalsensor, or others suitable device.

In another aspect, the invention relates to a method of marking aspecimen. The method includes the steps of positioning an object ofinterest within the specimen to a marking position, contacting thespecimen with a marker, and actuating the specimen to create indiciathereon at least partially bounding the object of interest. The shape ofthe indicia can be line segments, arcs, and combinations thereof. In oneembodiment, the specimen is disposed on a motorized stage and the methodincludes the step of actuating the motorized stage to position a capmechanism into an engagement position for sealing the marker.

In yet another aspect, the invention relates to an automated method ofreviewing a specimen. The method includes the steps of: (a) loading thespecimen in an optical instrument; (b) locating a first datum mark onthe specimen; (c) locating a second datum mark on the specimen; (d)establishing a coordinate system based, at least in part, on the firstand second datum marks; (e) positioning the specimen to present a firstfield of interest; (f) moving to a next field of interest; (g) repeatingstep (f) until a predetermined number of fields of interest arepresented; and (h) performing an autoscan of the entire specimen. In oneembodiment, twenty-two fields of interest are presented. In someembodiments, the step of moving to a next field of interest includesinputting a user command.

In various embodiments, the method can include the step ofelectronically marking an object of interest located within the field ofinterest, which includes inputting a signal to a processor, determiningif coordinate values for the object of interest are stored as a targetzone within the processor, and adding the coordinate values of theobject of interest to a list of marked target zones, if not previouslystored. Alternatively, the step of electronically marking the object ofinterest includes inputting a signal to a processor, determining ifcoordinate values for the object of interest are stored as a target zonewithin the processor, and removing the coordinate values of the objectof interest from a list of marked target zones, if the values arepreviously stored. The method can also include the step of indicatingvisually a marked status of the object of interest.

In addition, the method can include the step of physically marking anelectronically marked object of interest, which includes inputting asignal to a processor, positioning the object of interest to a markingposition, contacting the specimen with a marker, and actuating thespecimen to create indicia thereon at least partially bounding theobject of interest. Further, the step of performing the autoscan can beperformed after all fields of interest are presented. The step ofphysically marking the specimen takes place after the autoscan isperformed.

In some embodiments, the step of establishing a coordinate systemincludes centering the first datum mark, assigning a referencecoordinate value to the first datum mark, storing in memory the firstdatum mark coordinate value, centering the second datum mark, assigninga reference coordinate value to the second datum mark, storing in memorythe second datum mark coordinate value, and performing a coordinatetransformation. The step of establishing a coordinate system can furtherinclude comparing the datum mark coordinate values to known,predetermined values to determine if the specimen is loaded in a properorientation.

In other embodiments, the method can include the steps of reading aspecimen identifier, accessing a specimen data record stored within thecomputer to obtain coordinate values for the object of interest, andupdating the specimen data record to include the coordinate values ofthe electronically marked objects of interest. The method can furtherinclude the step of calibrating the marker prior to marking thespecimen.

In some embodiments, the step of calibrating the marker includesfocusing on an indicia disposed on the specimen, securing a marker intoa holder coupled to the optical system, contacting the specimen with themarker, actuating the specimen to create a calibration mark thereon,creating an offset value for the marker by actuating the stage toposition the calibration mark into a position relative to the indicia,recording the offset value, and applying the offset value to the markerposition for future marks. Further embodiments include the marking stepbeing performed after the entire specimen is scanned. Also, the autoscanstep maybe performed after all fields of interest have been presented.The specimen could be cytological material disposed on a slide that isstained with a thionin-phenol solution. The phenol can be a phenolderivative.

In another aspect, the invention relates to a method of accommodatingfor vibration errors in an imaging system. The method includes the stepsof acquiring vibration measurements of the imaging system while scanningportions of a specimen and re-imaging a portion of the specimen when thevibration measurement for the portion exceeded a predeterminedthreshold. In some embodiments, the method includes the step ofrejecting the specimen if the vibration measurement for a predeterminednumber of portions exceeds the threshold or the step of rejecting thespecimen if the vibration measurement exceeds a second threshold. In oneembodiment, the vibration can be measured by an accelerometer disposedon the imaging system.

In another aspect, the present invention relates to a slide holderassembly that can be used with an imaging system. The assembly includesa base, a first platform, a second platform, at least one slidepositioning member connected to the second platform, and an actuatingmechanism attached to the base for moving the slide positioning memberinto and out of contact with a slide. The first platform is movablydisposed on the base. The second platform is disposed on the firstplatform and includes a slide receiving area. The slide positioningmember is operatively connected to the second platform. The actuatingmechanism is disposed on the base. In various embodiments of theinvention, the base can be coupled with an actuating table, and theactuating table can be coupled to an imaging system.

In some embodiments, the assembly can include two slide positioningmembers operatively connected to the second platform. The mechanism foractuating the slide positioning member can include a first a pin and asecond pin for actuating the first and second slide positioning members,respectively. In other embodiments, the assembly can include at leastone stop disposed on the second platform. The slide positioning memberscan each be connected to a resilient member to bias the slidepositioning members towards the slide receiving area of the secondplatform. The slide positioning members can be rotatably mounted to thesecond platform and can position a slide in the receiving area into acontact position with the stop. In other embodiments, at least two stopsare disposed on the second platform.

The slide positioning members themselves can be selected based on theirmaterial properties and then mounted on the slide holder assembly suchthat they are actuated independently of one another in variousembodiments of the invention. The slide positioning members can beserially actuated with respect to one another. In addition, the slidepositioning members can be actuated orthogonally with respect to oneanother. The positioning members can each include a substantiallyelongate arm with a mounting end and a slide contacting end.

According to other embodiments of the invention, the second platform caninclude a sensor disposed in the slide receiving area for detecting thepresence of a slide. This sensor may be a hall-effect switch, aproximity switch, an optical sensor, a limit switch, or any othersuitable sensor.

In other embodiments of the invention, the slide holder assembly caninclude a slide with a cytological specimen, where the specimen islocated in a receiving area of the slide. This specimen can be stainedwith a conventional stain or a thionin-phenol stain solution. Thethionin-phenol solution can include a phenol derivative.

According to an aspect of the invention, the automatic focusing andimaging of locations of specimens on sample slides allows thoroughanalysis of sample slides, such as biological or cytological sampleslides, at accuracies and speeds much greater than would otherwise bepossible. During the analysis of a slide, it is routinely necessary toreadjust focus at various locations on the slide. Systems that aredesigned to automatically image substantially all of a region ofinterest of a slide need to enable the automatic adjustment of focus forvarious locations within that region of interest. A system which runsthrough a complete autofocusing procedure at every position it imagesmay accurately image substantially all of a region of interest, but itis inefficient, since many of the autofocusing steps taken have beendetermined to be redundant and therefore unnecessary. Accordingly, thisinvention provides an automatic focusing method for an optical systemthat efficiently and accurately images a series of regions on the slideto address efficiently substantially all of the area of interest of theslide.

In one aspect, the invention relates to an automatic focusing method foran optical system that provides a way to efficiently and accuratelyimage a series of regions on a specimen slide and cover substantiallyall of the areas of the specimen. One embodiment of the inventioninvolves a method for establishing a global focal plane for the sampleslide in order to account for tilt of the slide due to deviations frompure normality to the optical path of the imaging optics. This can occurdue to normal assembly tolerances of the slide holder, optical systemwear, and slide loading or holding misalignment. In establishing theglobal focal plane, another embodiment of the invention provides a checkof the slope along an index axis and the slope along a scan axis of theslide, to determine whether a predetermined threshold is exceeded. Inthis embodiment, if the threshold is exceeded, the slide is flagged toindicate the problem and appropriate corrective action can be taken.

Another embodiment of the invention involves a method of performing ascan pass across the surface of a slide in which a coordinate isdetermined that provides a focus value within a predetermined range ofan optimal focus value for a position on the slide. The coordinate isrecorded, and may be later retrieved. This coordinate may be expressed,for example, in a Cartesian coordinate system as the focus axiscoordinate or z-coordinate, corresponding to the (x,y) point thatcorresponds to a respective position on the sample slide in a plane ofthe sample slide. It should be noted, however, that the invention alsoapplies to configurations other than those involving a planar sampleslide and may be applied using coordinate systems other than theCartesian coordinate system, such as biradial, spherical, or cylindricalcoordinate systems, among others.

Another embodiment of the invention involves a method of determining anarea of fine focus jurisdiction around a position on a slide at which acorresponding value of a coordinate that provides a focus value within apredetermined range of an optimal focus value has been determined. Forinstance, once a z-coordinate has been determined by successiveadjustments of an element of the optical system imaging a position onthe slide, the same z-coordinate will provide adequately in-focus imagesfor positions in a given area surrounding that point on the slide. Anembodiment of the invention provides that an area of fine focusjurisdiction is elliptical in shape. Another embodiment provides that anarea of fine focus jurisdiction has a major axis substantially parallelto a scan axis of the optical system and a minor axis substantiallyparallel to an index axis of the optical system.

Another embodiment of the invention involves the determination of asecond coordinate that provides a focus value within a predeterminedrange of an optimal focus value for a second position on a slide. Thisembodiment determines whether the second point lies within at least onepreviously-determined area of fine focus jurisdiction, and if not,determines an area of fine focus jurisdiction surrounding the secondpoint. This embodiment optionally additionally includes the performanceof a fine focus action to determine a coordinate providing sufficientfocus at the second position.

Another embodiment provides for the use of a global focal surface of theslide in determining a first estimate of a coordinate providing adequatefocus. For example, tilt of the slide may be accounted for in estimatinga suitable focus axis coordinate.

Yet another embodiment provides for the determination of whether asecond point lies within at least one area of fine focus jurisdictionand the retrieval of the coordinate(s) correlated with this/thesearea(s). For example, if one or more previously-determined z-coordinatesapply over a certain region of a slide, the method will retrieve thesecoordinates instead of performing a new fine focus action, therebysaving the time associated with performing unnecessary steps. Anadditional embodiment provides for the adjustment of a retrieved valueaccording to the global focal surface of the slide.

Yet still another embodiment obtains a weighted average of retrievedcoordinates for a position on the slide corresponding to a point lyingwithin more than one fine focus jurisdiction. An additional embodimentprovides for the adjustment of this value according to the global focalsurface of the slide.

Another embodiment searches for previously-measured coordinate valuesonly from among those within a bin wherein a given point lies. In thisway, the process may be made more efficient. Still another embodimentdetermines whether or not a given position is within an area of interestof a slide. Another embodiment combines elements of the embodimentsdescribed above in various combinations and permutations andsystematically images substantially the entire area of interest of aslide in a step-by-step fashion.

Still another embodiment provides for an automatic focusing methodinvolving two kinds of focus actions—an initial coarse focus andsubsequent fine focuses. The initial coarse action is a more thoroughprocedure than the fine focus actions and may provide a baselinecoordinate used to enable quicker performance of the subsequent finefocus actions. Another embodiment limits the number of times the slideis imaged at a particular position to five.

These and other objects, along with advantages and features of thepresent invention herein disclosed, will become apparent throughreference to the following description of embodiments of the invention,the accompanying drawings, and the claims. Furthermore, it is to beunderstood that the features of the various embodiments described hereinare not mutually exclusive and can exist in various combinations andpermutations.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention. In the followingdescription, various embodiments of the present invention are describedwith reference to the following drawings, in which:

FIG. 1 is a graphical representation of one embodiment of a reviewsystem in accordance with the invention;

FIG. 2 is a flow chart illustrating the operational modes of the reviewsystem of FIG. 1;

FIG. 3 is a flow chart illustrating the operational mode of loading aslide into a review system;

FIG. 4 is a perspective view of a review system including a reviewstation, user interface, and a console;

FIG. 5A is a perspective front view of the review station of FIG. 4;

FIG. 5B is a perspective top view of the review station of FIG. 4;

FIG. 5C is a schematic front view of the review station of FIG. 4;

FIG. 5D is a schematic side view of the review station of FIG. 4;

FIG. 5E is a schematic cross-sectional view of the review station ofFIG. 4 taken at line 5E-5E in FIG. 5C;

FIG. 6 is a perspective view of the user interface shown in FIG. 4;

FIG. 7 is a perspective view of the console shown in FIG. 4;

FIG. 8 is a perspective view of one embodiment of a marker module foruse with a review system in accordance with the invention;

FIG. 9 is an exploded perspective view of the marker module of FIG. 8;

FIG. 10 is a perspective view of a mark indicator module for use with areview station in accordance with the invention;

FIG. 11 is a schematic cross-sectional view of the mark indicator moduleof FIG. 10 taken at line 11-11;

FIG. 12 is an exploded perspective view of the mark indicator module ofFIG. 10;

FIG. 13 is an exploded perspective view of a motorized slide stage;

FIG. 14 is a schematic view of one embodiment of a physical mark;

FIG. 15A is a schematic view of a specimen slide after being reviewed ona review system in accordance with the invention;

FIG. 15B is an enlarged view of a section of the specimen slide of FIG.15A;

FIG. 16 is a schematic view of one embodiment of a mark indicator;

FIG. 17 is an electrical schematic of a review station in accordancewith the invention;

FIG. 18A is a schematic view of one embodiment of an accelerometer;

FIG. 18B is a schematic view of an imaging system structure with theaccelerometer of FIG. 18A mounted thereon;

FIG. 19 is an exploded perspective view of one embodiment of a slideholder in accordance with the invention;

FIG. 20 is an exploded bottom perspective view of another embodiment ofthe slide holder in accordance with the invention;

FIG. 21 is a schematic top view of the slide holder of FIG. 19;

FIG. 22 is a schematic top view of the slide holder of FIG. 21 withslide positioning members securing a slide against two stops;

FIG. 23 is a perspective view of the slide holder of FIG. 20;

FIG. 24 is an exploded perspective view of one embodiment of a secondplatform for use with a slide holder in accordance with the invention;

FIG. 25 is a partial schematic top view of the slide holder of FIG. 19including a slide;

FIGS. 26A and 26B are perspective views of two embodiments of slidepositioning members;

FIG. 27 is a schematic diagram illustrating various manufacturingtolerances for one embodiment of a float glass slide for use in thepresent invention;

FIG. 28 is a schematic representation of a pattern of fine focusjurisdictions across a cell spot in accordance with the invention;

FIG. 29 is a schematic representation of a global focal surfacedetermination flowchart summarizing certain process steps in accordancewith the invention;

FIG. 30 is a schematic representation of a sample plot of focus axisposition versus focus score in accordance with the invention;

FIG. 31 is a schematic representation of a scan pass flowchartsummarizing certain process steps in accordance with the invention; and

FIG. 32 is a schematic representation of an area of fine focusjurisdiction in accordance with the invention.

DETAILED DESCRIPTION

Embodiments of the present invention are described below. It is,however, expressly noted that the present invention is not limited tothese embodiments, but rather the intention is that modifications thatare apparent to the person skilled in the art and equivalents thereofare also included.

The review system (“RS”) defines apparatus used by a cytotechnologist orcytopathologist (collectively “user”) to view a slide having acytological specimen disposed thereon. The review can be through eithera customized optical instrument or a traditional microscope interfacethat utilizes automatic slide movement. The automatic movement presentsfields of interest identified by an imaging system. Additionally, thereview system provides a method for automated marking of objects forlater review. The marking may be electrical, physical, or both.

Generally, when a user places a specimen on the RS for review, they arepresented a plurality of fields of interest (“FOIs”) that werepreviously identified by an imaging system and stored in a computerserver. In one operational state, the RS requires that each of theidentified FOIs be presented to the user before the user can completethe review of the specimen. As each of the FOIs is being presented, theuser can electronically mark the contents of a target zone within an FOIfor subsequent physical marking. Upon completing the electronic markingprocess, the RS can physically mark each electronic location (markedtarget zone) with a translucent marking dye. The actual details ofmarking are discussed hereinbelow. An electronic file representing thespecimen details and provided earlier by the server is used and updatedby the RS and maintained on the server.

The RS subsystem architecture is shown graphically in FIG. 1, whichillustrates the functional breakdown of the elements in the RS 1 intosubsystems. Generally, the elements of the RS 1 include a review station2, a user interface 4, a console 6, and a processor 8, all of which aredescribed in greater detail hereinbelow. The elements are in electrical,mechanical, or data communication with one another. In some instances,the elements are in a combination of electrical, mechanical, and datacommunication.

FIG. 2 illustrates the relationship of the various RS operational modesand FIG. 3 illustrates the functions that the RS 1 performs during theload specimen mode 606. Referring generally to FIG. 2, the functionalaspects of the various operational modes will be described. There are anumber of functions that are enabled in more than one mode and functionidentically. The various modes include: power off 600, pre-systemconnect 602, user login 604, load specimen 606, register specimen 608,autolocate 610, review 612, autoscan 614, 622, mark specimen 616, checkregistration 618, and unload specimen 620. The power off mode 600 iswhen there is no power applied to the RS 1. Upon the application ofpower, the RS 1 transitions to the pre-system connect mode 602. Thepre-system connect mode 602 represents RS operation prior to connectionto the server. The pre-system connect mode 602 includes a power on selftest (“POST”).

The POST functions include ram check, processor check, audible/visualindicator tests, console/user interface presence test indicating to theuser via the console that the RS 1 is initializing stage motor homingsequence, nosepiece motor homing sequence, object marker test, andspecimen ID reader test. If the POST is successful (all elements pass),the RS 1 initiates connection with the server; however, if the POST isunsuccessful, the RS 1 transitions to an error handling mode 632. If theserver connection is successful, the RS 1 can download lab preferencesfrom the server and transition to the user login mode 604. If, after apredetermined time, the server connection is still unsuccessful, the RS1 can warn the user that the server connection was unsuccessful andprompt the user to enter a manual review mode 636 or try to re-establishconnection with the server. If the user selects ‘re-try’, the RS 1reenters the pre-system connect mode 602. If the user selects manualreview mode 636, the RS 1 moves the specimen to a load position, promptsthe user to load a specimen and confirms by inputting a signal, andtransitions into manual review mode 636 upon receipt of an input signal.

During the user login mode 604, the user logs into the RS 1 and theuser's preferences are downloaded from the server. Specifically, the RS1 can prompt the user to enter a login ID via a keypad and press theenter key. During user ID entry, the RS 1 can display the ID in hiddenformat (placeholders only) and the backspace key can function as asingle character delete. After the user presses the enter key followingID entry, the RS 1 queries the server database and, if the ID exists,set user preferences locally according to the server response andtransition to the load specimen mode 606. If the ID does not exist, theRS 1 emits an audible tone, warns the user (“Invalid ID”) via thedisplay, delays and then returns to user login mode 604. If the RS 1receives a broadcast message from the server informing it of pendingsoftware update activity, the RS 1 can return a response to the messageand transition to a software update mode 638. If the RS 1 receives abroadcast message from the server informing it of pending systemdiagnostic activity, the RS 1 can return a response to the message andtransition to a system diagnostic mode 624, 634.

The load specimen mode 606 (FIG. 3) begins with the RS 1 waiting for theuser to load a specimen. The specimen ID is read automatically by the IDreader or input manually from the keypad if the automatic read fails. Ifthe ID corresponds to a valid record on the server corresponding to animaged specimen, the RS 1 proceeds with registration, otherwise the RS 1continues with manual review. The RS 1 can enable the cancel key on theconsole during manual slide ID entry. The RS 1 can also enable aspecimen presence sensor. The RS 1 can move the stage to the specimenload position and prompt the user to load a slide and input a signal viathe user interface to confirm the start of an automated slide review orstart manual review. Upon receipt of the input signal confirming thestart of automated slide review, the RS 1 can move the stage such thatthe specimen ID is positioned under the specimen ID reader. The RS 1then sends a message to the specimen ID reader to initiate theillumination of the specimen label, as well as the capture andprocessing of an image. The RS 1 interprets the specimen ID stringreceived. If the specimen ID has a valid format/checksum, operationcontinues with the RS 1 querying the server for existence of a specimendata record with this slide ID. If there was a “bad read” or if theformat/checksum are invalid, the RS 1 displays an invalid slide IDmessage, issues an audible tone, and waits for the user to press thecontinue key on the console.

Upon receipt of the continue instruction, the user manually enters thespecimen ID (including checksum) via the console or cancels the specimenreview. During manual specimen ID entry, the RS 1 can display thespecimen ID. During manual specimen ID entry, the backspace key canfunction as a single character delete. Upon receipt of the specimen ID,the RS 1 can interpret the manually entered specimen ID. If the specimenID has a valid format/checksum, operation resumes with the RS 1 queryingthe server for existence of a specimen data record with this slide ID.If the format or checksum is invalid, the RS 1 displays an invalidspecimen ID message and issues an audible tone.

Once operation resumes, the RS 1 queries the server for existence of aspecimen data record with the corresponding specimen ID. If the serverresponse to the database query indicates the presence of a specimen datarecord (“SDR”) corresponding to this specimen ID, the RS 1 downloads thenumber of fields of interest (“FOIs”), the locations (in Image Processor(“IP”) coordinates) of the FOIs, the locations (in IP coordinates) ofthe fiducial marks, the locations (in IP coordinates) of the markedtarget zones from previous reviews (if any), the location (in IPcoordinates) of the specimen cell spot center, the specimen cell spotdiameter, and the specimen reviewed status. If the server response tothe database query indicates a communication error or that there is noslide data record corresponding to this slide ID, the RS 1 can issue anappropriate warning on the console, emit an audible tone, and instructthe user to press the continue key to cancel slide review.

After the RS 1 has downloaded the specimen data record, the RS 1 checksto see if the specimen has already been reviewed. If the specimen waspreviously reviewed, the RS 1 will display a warning message, emit anaudible tone, inhibit the unmarking of target zones marked in previousreviews, and request confirmation that the user wishes to proceed. TheRS 1 waits for this confirmation, then transitions to the register slidemode. If the specimen was not previously reviewed, the RS 1 transitionsdirectly to the register specimen mode 608.

In the register specimen mode 608, the RS 1 establishes the registrationof the specimen by having the user center each of two fiducial marks inan area cradled by a mark indicator in the field of view (“FOV”). Havingthe coordinates of the fiducial marks in both IP (from the SDR) andreview station systems, the RS 1 then computes and performs a coordinatetransformation on the field of interest (“FOI”) data obtained from theserver. The specimen presence sensor is enabled during the specimenregistration mode. The RS 1 illuminates a white mark indicator andpresents the first fiducial mark within the FOV with the objective at10×. The RS 1 then enables the specimen position input device (“SPID”)for manual stage positioning limited to about plus or minus half an FOV(e.g., 1100 μm) along either of the two slide stage motion axes (x, y).The RS 1 prompts the user to use the SPID to center the first fiducialmark within the mark indicator and enter a next command when theoperation is complete. Upon confirmation, the RS 1 presents the secondfiducial mark (position derived from the SDR plus the offset recorded incentering the first fiducial mark) and prompt the user to use the SPIDto center it within the mark indicator and enter the next command whenthe operation is complete or enter a previous command to go back to thefirst fiducial mark. Upon receipt of the previous command, the RS 1returns to the location of the user-centered first fiducial mark. Uponconfirmation that the alignment of the second fiducial mark is complete,the RS 1 computes a coordinate transformation from IP to review stationbased upon the fiducial mark coordinates as recorded in the SDR and aslocated at the review station by the user. The RS 1 also applies thecoordinate transformation to each FOI location, marked target zone, andthe specimen cell spot center, and then transitions into autolocate mode610.

In the autolocate mode 610, the user is presented with a predeterminednumber of FOIs using the 10× objective. At any time while in this mode,the user may mark/unmark target zones, manually move the stage using theSPID, and toggle the objective lens. Autolocate is complete when allFOIs have been presented. On the initial review of the specimen,autolocate is typically completed before the user is permitted to reviewmarked target zones, perform an autoscan or complete specimen review.

During the autolocate mode 610, the RS 1 indicates the current mode andprogress, (e.g., “1 of 30”) on the display and instructs the user (viathe display) on how to transition to the next or previous FOI. The RS 1presents the first FOI in the FOV and enables the SPID for user-directedpositioning of the stage. The RS 1 enables electronic marking/unmarkingand mark indication while the objective is at 10× The RS 1 also enableschanging of magnification and the specimen presence sensor.

The next and previous inputs on the user interface allow the user tochange the objective lens to 10×, if not already at 10×, position thestage at the next or previous FOI, update the autolocate mode progressindicator (e.g., “3 of 30”) on the display, and instruct the user (viathe display) on how to transition to the next or previous FOI. If theuser enters next when the current FOI is the last FOI (e.g., all FOIshave been presented), the RS 1 emits an audible tone and indicates onthe display, as part of the status of specimen review, that autolocateis complete, if this has not already been done. The RS 1 ignores aprevious command if the present FOI is the first FOI. The previouscommand may be accompanied by a warning, such as a an audible tone.

If the user commands a change to the review mode 612 (e.g., via a reviewkey on the console), the RS 1 changes the objective lens to 10× (ifnecessary) and transitions into review mode 612. In one embodiment, auser can only change to review mode 612 if the number of marked targetzones is greater than zero and the initial autolocate review of thespecimen has been completed. If the user commands a change to theautoscan mode 614, 622 (e.g., via a scan key), the RS 1 changes theobjective lens to 10× (if necessary) and transitions into autoscan modepaused 614. In one embodiment, autoscan can only be selected on theinitial review of the specimen and only if the autolocate mode 610 iscomplete. If the user enters a finish command during the initial reviewof the specimen, the RS 1 changes the objective lens to 10× (ifnecessary) and transitions to the check registration mode 618. During asubsequent review of the specimen, when there are new electronic marks,the RS 1, in response to the finish command, changes the objective lensto 10× (if necessary) and transition to the mark specimen mode 616. Ifduring a subsequent review of the specimen, when there are no newelectronic marks, the RS 1, in response to the finish command, changesthe objective lens to 10× (if necessary) and transitions to the checkregistration mode 618.

In the review mode 612, electronically marked target zones from thecurrent specimen review, as well as marked target zones from previousreviews, are presented to the user using the 10× objective. The RS 1indicates the current mode and progress on the display, instructs theuser (via the display) on how to transition to next/previous markedtarget zones, and presents the first marked target zone (from the firstslide review if this is a subsequent review) in the FOV. In addition,the RS 1 enables the SPID for user-directed positioning of the stage,electronic marking/unmarking while the objective is at 10×, markindication while the objective is at 10×, and changing of magnification.

During the review mode 612, all the electronically marked target zones,including any marks from previous slide reviews, are available forpresentation. The order of presentation of marked target zones may be,for example, chronological, such as in the order target zones weremarked. Alternatively, the presentation order of the marks may begeographic based. When the user enters a next or previous command viathe user interface, the RS 1 changes the objective lens to 10×, if notalready at 10×, positions the stage at the next or previous(respectively) marked target zone, updates the review mode progressindicator (e.g., “2 of 5”) on the display, and instructs the user on howto transition to a next or previous marked target zone. The RS 1 ignoresthe next command if the present marked target zone is the last markedtarget zone. The RS 1 also ignores the previous command if the presentmarked target zone is the first marked target zone. The RS 1 maygenerate a warning, such as a an audible tone.

If the user commands a change to the autoscan mode 614, 622, the RS 1moves to the scan start position if an autoscan has not yet beeninitiated or moves to the current scan position if an autoscan hasalready been initiated. The RS 1 subsequently transitions into autoscanpaused mode 614. Typically, the user may only command a change to theautoscan mode 614, 622 during an initial specimen review and only if theautoscan has not been completed. If the user enters a finish commandduring the initial review of the specimen, the RS 1 changes theobjective to 10× (if necessary) and transitions to the mark specimenmode 616. If the user enters a finish command during a subsequent reviewof the specimen and there are new electronic marks, the RS 1 changes theobjective to 10× (if necessary) and transitions to the mark specimenmode 616. If the user enters a finish command during a subsequent reviewof the specimen and there are no new electronic marks, the RS 1 changesthe objective to 10× (if necessary) and transitions to the checkregistration mode 618.

Autoscan mode 614, 622 implements an automated full scan of the specimencell spot with the 10× objective in place. Autoscan may be automatic oruser-triggered. In an automatic autoscan, motion is initiated by the RS1 and may be paused and resumed by the user. While paused, the user canuse the SPID for manual positioning and mark/unmark as in autolocate andreview modes 610, 612. The scan resumes where it left off when the userenters a next or resume command. The motion profile for an automaticautoscan may be continuous, consisting of a series of overlappingscan-lines (during which the scan speed can be controlled dynamically bythe SPID) or intermittent (i.e., start-stop), consisting of a series ofmovements to discrete, overlapping FOVs and including a pause at eachFOV, the duration of which can be controlled dynamically by the SPID.

The user-triggered autoscan is identical to a start-stop automaticautoscan, except that the user initiates the transitions to successiveFOIs by entering a next or previous command. In addition, the autoscandirection can be horizontal or vertical, according to the user'spreference. The autoscan is complete when the entire sequence of movesto scan line endpoints (or discrete FOV) has been completed. In oneembodiment, a first review of a specimen requires the entire autoscan tobe completed if there are any electronic marks. Generally, the autoscanshould cover 100% of the specimen cell spot with overlap between FOVs.

At the start of the autoscan mode 614, 622, the RS 1 calculates the endpoints of all the moves that constitute the scan based upon the cellspot center from the SDR and the selected motion profile (start-stop orcontinuous) and orientation (horizontal or vertical). The RS 1 moves tothe scan start position. In addition, the RS 1 indicates autoscanprogress and paused condition on the display (e.g. “scan paused—25%complete”), which will prompt the user to enter the next or resumecommand to begin/resume the scan if the scan is not complete. The RS 1also enables the SPID for user-directed positioning of the stage, theelectronic marking/unmarking function while the objective is at 10×, themark indication function while the objective is at 10×, and the changingof magnification. If the user enters a reset command during autoscan,the RS 1 moves to the scan start position and cancels the scan inprogress.

If the user enters the previous command during automatic start-stopautoscan, the RS 1 backs the stage up one FOI, if the current scanposition is not the scan start position and the scan is not complete.When the user enters a next or resume command during a user-triggeredautoscan and the scan is not complete, the RS 1 moves to the next FOIand updates autoscan progress on the display. When the user enters thenext or resume command and the scan are complete, the RS 1 emits anaudible tone. When the user enters a pause or previous command and thecurrent scan position is not the scan start position, the RS 1 moves tothe previous FOI and updates the autoscan progress on the display. Whenthe user enters the pause or previous command and the current scanposition is the scan start position, the RS 1 emits an audible tone.

If the user commands a change to the review mode 612 when the number ofmarked target zones is greater than zero, the RS 1 changes the objectiveto 10× (if necessary), stores the current scan position, and transitionsinto review mode 612. If there are marks and the user enters the finishcommand, the RS 1 transitions to the mark specimen mode 616. If thereare no marks and the user enters the finish command, the RS 1transitions to the check registration mode 618.

When the user enters a next or resume command during an automaticautoscan and the scan is not complete, the RS 1 changes the objectivelens to 10× (if necessary), moves the stage to the current stored scanposition (in case user-directed positioning occurred), and transitionsto an autoscan in progress mode 622. Autoscan in progress mode 622 iswhen automatic scanning is underway and operation has not been paused bythe user. It is typically only entered during an automatic (continuousor start-stop) autoscan.

During a continuous autoscan, the RS 1 moves to the end point of thefirst (present) scan line at a speed defined by the user. When theX-axis of the SPID is deflected to the right, outside a pre-defineddeadband zone, the RS 1 continuously adjusts the scan speed upward.Conversely, when the X-axis of the SPID is deflected to the left, the RS1 adjusts the scan speed downward. After completion of each scan line,the RS 1 updates the autoscan progress indicator on the display.

During a start-stop autoscan, the RS 1 moves to the first (next) FOI ata speed defined by the user and waits there for a dwell time defined bythe user. When the X-axis of the SPID is deflected to the right, outsidea pre-defined deadband zone, the RS 1 continuously adjusts the dwelltime downward. Conversely, when the X-axis of the SPID is deflected tothe left, the RS 1 adjusts the dwell time upward. After arrival at eachFOI, the RS 1 updates the autoscan progress indicator on the display.

During either a continuous scan or a stop-start scan, the velocity ofinter-scan line moves is at the FOI-to-FOI speed defined by the user.The RS 1 continues moving to FOIs until the final scan line end point orFOI is reached, at which time the RS 1 transitions to autoscan pausedmode 614. When the user enters a pause or previous command prior to scancompletion, the RS 1 pauses stage motion, stores the current scanposition, and transitions to autoscan paused mode 614.

In the mark specimen mode 616, physical marks are made on the specimenin batch fashion by an object marker at the locations of the electronicmarks made during the present specimen review. The physical marks arethen individually presented to and verified by the user. The markindicator cursor illuminates green to indicate that electronicallymarked target zones exist. Upon entering the mark specimen mode 616, theRS 1 translates the electronically marked target zone coordinates intoobject marker coordinates using the current translation values for theobject marker obtained during the most recent object marker calibration.For each target zone electronically marked during the current reviewsession (typically in the order in which it was electronically marked)the RS 1 positions the stage (as necessary) for marking, and createsstage motion (as necessary) and marker motion sufficient to make thephysical mark on the specimen. For each target zone physically markedduring the current review session, the RS 1 presents the physical markwith the objective at 10× and prompts the user to enter a next commandto verify the mark or enter a reject command if it is not verifiable.

If the user enters reject for any mark, the RS 1 prompts the user toconfirm that the mark is to be rejected by inputting a yes or nocommand, which typically involves pressing the ‘Y’ or ‘N’ key on thekeypad. Upon receipt of the yes command, the RS 1 indicates via thedisplay that the mark has been rejected, emits an audible tone, andwaits for the user to enter a continue command, after which the RS 1transitions to the load specimen mode 606. In one embodiment, the RS 1prompts the user to erase all physical marks. Upon receipt of the nocommand, the RS 1 restores the prior operational state. When the userverifies the last of the physical marks, typically by entering a nextcommand, the RS 1 caps the marker, if the marker was equipped with acap, and transitions to the load specimen mode 606 if this is a manualreview and to the check registration mode 618 otherwise.

If the user enters a cancel review command, the RS 1 transitions to themanual review mode 636 accompanied by a warning, for example, suggestingthat the user calibrate the marking device and/or an audible tone. Whenmarks are present, the RS 1 additionally prompts the user to erase allphysical marks. When the user verifies the last of the physical marks,the RS 1 transitions to manual review mode 636.

In the check registration mode 618, the registration that wasestablished at the beginning of the specimen review in the registerspecimen mode 608 is verified. Verification of registration confirmsaccurate presentation of all FOIs, as long as the specimen did not moveand later recover position during specimen review. The RS 1 illuminatesthe white mark indicator cursor. For each of the two fiducial marks, theRS 1 positions the stage at the position the user indicated in theregister specimen mode 608 with the objective at 10× and prompts theuser to confirm the alignment of the first fiducial mark in the targetzone by entering the next command or reject registration by entering thereject command.

If the user enters the reject command after the presentation of eitherfiducial mark, the RS 1 displays a warning (e.g., mark rejected), emitsan audible tone, turns off the mark indicator, and/or prompts the userto enter the continue command. When marks are present on the initialreview, the RS 1 additionally prompts the user to erase all physicalmarks. After entry of the continue command, the RS 1 transitions to theload specimen mode 606. If registration is verified, which the user canindicate by, for example, entering the next command twice in succession,the RS 1 turns off the mark indicator and transitions to the unloadspecimen mode 620.

In the unload specimen mode 620, the RS 1 uploads the updated SDRinformation to the server and makes a specimen reviewed mark on thespecimen boundary arc. In addition, the RS 1 transforms the markedtarget zone locations from the present review (new marks) back into theIP coordinate system by reversing the transformation performed duringthe register specimen mode 608 and updates the SDR on the server withthe time of the start of specimen review, the total number of markedtarget zones (any new plus any old, and the new marked target zonelocations in IP coordinates), and the time of the completion of specimenreview (present time).

If the review was the first review of the specimen, the RS 1 positionsthe stage for creation of the specimen reviewed mark, uncaps the pen ormarker, creates the stage motion and the marker motion as necessary tomake the specimen reviewed mark on the specimen boundary arc, caps thepen, moves the stage to the specimen load position, emits an audibletone, and prompts the user to unload the specimen. When the specimen isremoved, as detected by a specimen presence sensor, the RS 1 transitionsto the load specimen mode 606.

Other operational modes include user logout 626, set user preferences628, error handling 632, marker calibration 640, software update 638,manual review 636, and diagnostics 624, 634. In the user logout mode626, the user logs out of the RS 1. The RS 1 prompts the user to confirmlog out, for example, by entering a yes or no command. If the userenters the yes command, the RS 1 uploads the user's preferences to theserver and transitions to the user login mode 604.

During the set user preferences mode 628, the user is able set heroperational preferences, including autoscan settings and maximum speedfor manual positioning. For each user-settable preference, the RS 1indicates to the user via the display the current setting of theparameter and how to change the setting to the desired value. Thesetting is stored locally until the user preferences are updated on theserver during the user logout mode 626. Other user settable preferencesinclude, but are not limited to, scan type, scan direction, autolocate,input setup, and SPID setup. By way of example only, the scan typesettings can include auto continuous, auto start-stop, or user-triggeredstart-stop; the scan direction can include left-right or up-down;autolocate settings can include the FOI-to-FOI speeds; and the SPIDsettings can include user-based or specimen-based.

At any time during the set user preferences mode 628, except whenrunning a test, if the cancel command is entered, the RS 1 transitionsto the load specimen mode 606. The RS 1 will not save any changes thatwere not followed by entering a save command. When running a test, ifthe cancel command is entered, the RS 1 stops all motion, cancels thetest, and returns to the menu displayed before entering the testcommand.

The error handling mode 632 is a special mode that handles errors withinthe RS 1. This mode can be entered from any mode except the power offmode 600. Within the error handling mode 632, the RS 1 assesses theerror and takes appropriate action. If the error does not prevent thesystem from continuing to operate, the RS 1 takes appropriate action andtransitions back to the appropriate mode.

In the marker calibration mode 640, the position of the mark created bythe marker is calibrated, for example, by being aligned with the stagecoordinate system. This procedure may be performed at a variety oftimes, for example, every time a new marker is installed, after an erroris encountered while verifying marks, or at least once per user session.During the marker calibration mode 640, the RS 1 prompts the user toload a blank specimen slide and confirms the slide is loaded by enteringa next command. Upon confirmation of slide load, the RS 1 makes a testmark on the slide at predetermined coordinates, presents the test markto the user using the 10× objective, and illuminates the white markindicator. Further, the RS 1 prompts the user to use the SPID to centerthe test mark within the mark indicator and confirm when the operationis complete. Upon confirmation of the test mark, the RS 1 generates thecoordinate translation values (ΔX, ΔY) or geometric offsets for themarker. The RS 1 then emits an audible tone, indicates completion ofmarker calibration on the display, and waits for the user to enter acontinue command, after which the RS 1 transitions to the load specimenmode 606.

The software update mode 638 enables the server to download and installnew software on the RS 1. All system functionality is typically lockedout until the software update is complete. Upon completion of thesoftware update, the RS 1 returns a message to the server andtransitions to the pre-system connect mode 602.

The manual review mode 636 allows the user to control the motorizedstage, the motorized nosepiece, and the marker. The user is able toelectronically mark/unmark and physically mark target zones, as well asreview the electronically marked target zones. Upon entry into themanual review mode 636, the RS 1 moves the stage to the specimen loadposition and prompts the user to load a specimen. After manuallyinputting a command to the RS 1, the stage moves to a nominal locationon the specimen for the start of the manual review.

During review, when the user enters a next or previous command, the RS 1changes the objective lens to 10×, if not already at 10×; positions thestage at the next or previous marked target zone, if any marked targetzones exist; emits an audible tone if no marked target zones exist or ifthe user enters the next command while at the last marked target zone orenters the previous command while at the first marked target zone; andupdates the marked target zone status indicator on the display.

If the user enters the finish command and there are electronic marks,the RS 1 transitions to the mark specimen mode 616. If the user entersthe finish command and there are no electronic marks, the RS 1transitions to the load specimen mode 606. In an alternate embodiment,opening the specimen holder causes the RS 1 to clear all electronicmarks and re-enter the manual review mode 636. When the usersubsequently enters a cancel command, the RS 1 clears all electronicmarks and re-enters the manual review mode 636. If the user enters afinish command and there are electronic marks, the RS 1 transitions tothe mark specimen mode 616. If the user enters the finish command andthere are no electronic marks, the RS 1 re-enters the manual review mode636.

A system diagnostics mode 628, 638 is entered when the RS 1 is in theuser login mode 604 and the server attempts to query the RS 1 forinformation. The RS 1 sends the requested information to the server andtransitions to the user login mode 604. The diagnostics mode does notallow for specimen review, but is used to test subsystem components andview the status of internal variables.

Some of the functions referenced hereinabove are now described ingreater detail. One such function is electronic marking and unmarking.When electronic marking is enabled, if the user enters a mark command,the RS 1 determines if the current location is electronically marked. Ifnot, the current X, Y coordinates are added to the list of marked targetzones. If the current location is already within a marked target zonemade during the current specimen review, the center coordinates of thetarget zone are removed from the list of marked target zones. Markindication is another function. When mark indication is enabled, the RS1 continuously checks stage position and determines if the currentlocation is within a marked target zone. If it is, the RS 1 illuminatesthe mark indicator to be green in color. Otherwise, the RS 1 illuminatesthe mark indicator to be white in color. The RS 1 performs this checkingduring user-directed positioning, while the stage is moving or whenmotion ceases. During change of magnification, one of the inputs on theuser interface is used to change objective lenses, for example, from 10×to 40× or from 40× to 10×. During cancel specimen review, if the cancelcommand is entered, the RS 1 stops all motion, stage and/or marker,emits an audible tone, and prompts the user for confirmation thatspecimen review is to be cancelled. If a yes command is entered, the RS1 transitions to the load specimen mode 606. The display may issue awarning (e.g., “erase all physical marks”). If a no command is entered,the RS 1 returns to its previous operational state. In addition, the RS1 may include a specimen presence sensor and associated functions. Ifthe specimen holder is opened at any time during an operational mode inwhich it is enabled, the RS 1 stops all motion, stage and/or marker,displays a warning (e.g., “specimen removed-review cancelled”), emits anaudible tone, and prompts the user to enter the continue command. If thespecimen holder is opened after the specimen has been physically marked,the display should additionally warn the user to erase all physicalmarks. Upon entering a continue command, the RS 1 restores the objectmarker to its resting state, if necessary, and transitions to the loadspecimen mode 606.

FIG. 4 depicts the RS 1 including peripherals. See also FIG. 1. The RS 1includes a review station 2, a user interface 4, and a console 6. The RS1 also includes a processor 8, which in FIG. 4 is located within thereview station 2. The review station 2, user interface 4, console 6, andprocessor 8 are in electrical, mechanical, and/or data communicationwith one another.

FIGS. 5A-5E are illustrations of the review station 2. In some of thefigures, components have been removed for clarity. The review station 2includes a frame 3, an optical instrument 10, an illuminator subsystem11, an illumination control panel 19, and focus controls 21. The reviewstation 2 also provides the structural and optical framework foradditional custom subsystems, which can be integrated into the reviewstation 2 for the purpose of providing the specific functionality. Morespecifically, the review station 2 includes a motorized stage 12, an OCRreader 16 (ID reader), a motorized two-position nosepiece 14, a markindicator 20 (visual indication of mark location in the FOV), an objectmarker 18 (physical marking of an object of interest in a specimen), aprocessor 8, a Koehler illuminator, an illuminator controller 23, anupper arm 27 (including the mark indicator module 20 and the objectmarker subsystem 18), and an eyepiece 29.

The frame 3 is similar in size and form as compared to microscopescurrently used in a clinical setting. The frame 3 has a lowcenter-of-gravity, resists damage due to shock or vibration, andminimizes potential pinch points, for example, from stage, nosepiece, orobject marker motion. In one embodiment, the frame 3 has a high degreeof stiffness to minimize movement in the eyepiece 29 during quick stagemoves. The frame 3 includes a upper arm 27 that has minimal deflection.In some embodiments, a support leg may be used to minimize further thedeflection of the upper arm 27. The FOV may become blurred during 40×viewing if the upper arm 27 deflects too much. The frame 3 canincorporate four rubber non-skid feet 37 fastened to its base. Theserubber feet 37 can minimize shock to the review station 2, dampenimpulses and environment oscillations, and prevent sliding on the benchtop surface. The frame 3 can be made from cast aluminum; however, othermaterials, as known to one of ordinary skill in the art, arecontemplated.

The review station 2 shown in FIGS. 5A-5E utilizes Koehler typeillumination for lighting the specimen. The illumination system providesthe user with easy access to the bulb chamber to replace the bulb andfor adequate thermal cooling in the illumination area. Proper coolingextends the bulb life and reduces user hazards related to the elevatedexternal surface temperatures. Adjustment of the illumination aperture41 (view diameter of the illumination source) has easy access, smoothmotion, and an adequate gripping surface. The illumination system 11includes an illumination control panel 19. The illumination controls(power, light intensity, green mark indicator intensity, and white markindicator intensity) are grouped within easy access of a focus knob 21.

The motorized nosepiece subsystem 14 is located on the bottom side ofthe upper arm 27 of the frame 3. The nosepiece 14 provides automatedselection and positioning of the objective lenses 43, 45 as commanded.In the embodiment shown in FIG. 5A, the objective lenses 43, 45 are 10×and 40×; however, other magnifications could be selected to suit aparticular application. The nosepiece 14 rotates from side-to-sideapproximately 60° to switch between the two objective positions. Thenecessary motor 13, sensors 17, and drive system are mounted behind theobjective holder 47. The right objective mount provides two set screwsto translate the objective in a plane perpendicular to the optical path.This allows for parcentricity adjustments. To provide parfocality,adjustment shims can be added between either of the two objectives 43,45 and the nosepiece 14 to decrease the distance to the common focalplane. The objective lenses 43, 45 can be standard Olympus UIS seriesinfinitely corrected optics. The nosepiece subsystem 14 operates using aDC permanent magnet brushed servomotor 13 with an integral gear head.The motor 13 operates at about 5 volts (V) and has about 22 ohms ofresistance. The gear head drives a cam follower that pushes theobjective holder 47 from side to side. Another cam and springarrangement causes the nosepiece 14 to toggle and hold over at correctobjective locations.

Electrically, there are three components, a motor 13 and two sensors 17,one for each end of travel, to indicate the objective is in place. Thetwo optical interrupt sensors 17 take +5V and ground inputs and producean output that is low when a flag is blocking the sensor. The motorizednosepiece 14 interfaces to the review station 2 via the processor 8. Thereview station processor 8 monitors the rotary position of the nosepiece14 via position sensors 17 connected to digital input ports. The usercan toggle the nosepiece position via a user interface input, such as anobjective selection button 15.

The specimen ID reader subsystem 16 is located within the upper arm 27of the frame 3. The slide ID reader 16 is used in the RS 1 to acquire animage of a specimen label and uses OCR to determine the specimen ID andCRC. In one embodiment, the specimen ID reader 16 is a custom WelchAllyn camera and processor that includes a camera, a lens, a CCD sensor,a light source, a processor, and a frame grabber. The specimen ID reader16 has a working distance (i.e., the distance from the camera lenssurface to the specimen label surface) of about 3.7 inches; however, theworking distance can vary to suit a particular application. The specimenID reader 16 has a viewing angle (i.e., the angle deviation fromparallel between the camera lens surface and the specimen surface) ofabout 13°+/−2°; however, the viewing angle can be chosen to suit aparticular application. The specimen ID reader 16 operates accurately inan external light environment such as normal office lighting (e.g., 100foot-candles).

The specimen ID reader 16 captures an image of the specimen ID and CRCon the specimen label. The specimen label may have at least one line ofcharacters with up to 7 characters on a line. The label may includecharacters representing a patient ID. In one embodiment, the last threecharacters are the CRC. All characters are OCR-A and are limited to thedigits 0 through 9 and the symbol “-”. The electrical interface to thespecimen ID reader 16 consists of two cable assemblies, which can becombined into one for routing from the review station processor 8.Communication over the interface is bi-directional and allows the reviewstation processor 8 to control the specimen ID reader 16 and receive thespecimen ID data or any error messages.

The motorized stage subsystem 12, which is located on the frame 3, canbe seen in FIG. 13. The motorized stage 12 moves the specimen in thehorizontal plane with respect to the motorized nosepiece 14 tofacilitate specimen loading, specimen ID reading, specimen review,viewing of the fiducial marks, and object marking. In addition, themotorized stage 12 is used to position the slide specimen area in theFOV to facilitate the location of FOIs, marked target zones, anddestinations arrived at manually. The stage 12 can move at a constantvelocity in a single direction within the cell spot boundary during anautoscan mode 614, 622. Further, the stage 12 moves the specimen tocreate physical marks, for example, the specimen reviewed mark. SeeFIGS. 15A and 15B. The stage 12 includes hard stops 61 to limit travelon the X and Y-axes 62, 64 and sensors 63 to indicate home positions,and a sensor 65 to sense the presence of a specimen on the stage 12. Thesensors 63, 65 are hall-effect switches, but could also be limitswitches, proximity switches, or optical or magnetic sensors. In oneembodiment, the stage size is about 255 mm wide, 280 mm deep, and 90 mmhigh.

The stage 12 provides travel along two orthogonal axes, where the X-axis62 is left/right and the Y-axis 64 is front/back. Each axis 62, 64includes a platform 69 guided on a linear bearing system. A DCservomotor 66 drives each axis 62, 64 through lead screws 68 with thedrive system using plastic anti-backlash nuts 70 to minimize backlash.In order to minimize loads on the motor shaft and maintain axialalignment of the encoder 72, the lead screws 68 are mounted with acentering coupling system 74 on the servomotor end and thrust bearingson the other end. Both axes 62, 64 are controlled using encoder feedbackand home sensors 63. The stage 12 includes a specimen holder 71 mountedon the Y-axis platform 69 to maintain alignment of the specimen with thestage 12. A spring-loaded arm 76 allows the user to load and unload thespecimen, and a sensor 65 is included to verify the presence of aspecimen in the specimen holder.

The stage 12 is attached to the frame 3 by a Z-axis horizontal mount.The stage 12 is fastened to the horizontal mount using screws; however,other commonly known fastening methods could be utilized, such aswelding, rivets, chemical bonding, and the like. The stage 12 includeslocating features, such as pins, in order to establish the stageposition relative to the frame 3. In one embodiment, the X-axis motionis about 60 mm and the Y-axis motion is about 56 mm. A double shaft, DCbrushed servomotor is used for the X and Y axis drive. The lead screwpitch is about 2 mm, and a self-centering coupling is used to connectthe lead screw to the motor to maintain axial alignment. A thrustbearing can be used to support the lead screw free end to minimizevelocity ripple. In one embodiment, the stage 12 has a maximum speed of32 mm/sec and a minimum speed of 0.02 m/sec; however, the stage speedcan be selected to suit a particular application. The drive connectionmay use an anti-backlash nut to minimize backlash, for example, a drivescrew/nut that is self-lubricating to eliminate servicing requirementsand minimize noise and stiction. The stage 12 incorporates motor covers77 to cover and protect the motors, sensors, and wire harnesses. Themotor covers 77 also protects the user from any possible pinch points.

The stage home position is determined using one sensor 63, for example ahall-effect switch, per axis. A hard stop 61 is used to prevent damagein the event of loss of motor control. The specimen holder 71incorporates a sensor 65, for example a hall-effect switch, to detectspecimen presence. In order for the specimen to be loaded properly andthe correct position maintained through a full sequence of operations,it is desired that the specimen sensor arm 79 actuation force besignificantly lower than the specimen retaining arm 76 actuation force.For example, the actuation force of the specimen sensor arm force can beabout 5 g to about 20 g and the specimen retaining arm force can beabout 100 g to about 140 g. This arrangement helps to prevent the sensorarm 79 from moving the specimen away from a datum position. The slideretaining arm 76 should apply sufficient force on the specimen toprevent any movement of the specimen away from the datum position duringobject marking. The specimen resting surface 73 is preferably stiff,flat, resistant to scratching, and has a low friction coefficient withlittle noticeable stiction. In one embodiment, the specimen restingsurface has a flatness of ±4 μm over 0.22 mm.

Each axis has a brushed DC servomotor 66 for motion. Each motor 66 canbe controlled via a separate servomotor interface module in datacommunication with the review station processor 8. Feedback is providedby an optical encoder 72 mounted on the motor shaft; however, otherfeedback devices can be used. The interface module supplies power to themotors 66 and monitors encoder feedback. Each axis has at least onesensor 63 that determines the home position of the stage 12 andinterfaces with the review station processor 8. The home sensors 63 areused to establish the stage home position. The specimen presence sensor65 also interfaces with the review station processor 8.

One embodiment of an illuminator subsystem in accordance with theinvention is shown in FIG. 5E. The illuminator subsystem 11 is locatedin the lower arm 49 of the frame 3 and provides for the illumination ofthe specimens being viewed on the review station 2. The frame 3 supportsand aligns the illuminator II with respect to the condenser assembly 57and optics. Alignment and calibration of the condenser assembly 57facilitates proper specimen illumination. The illuminator II includes abase plate with access door, a power supply, an illumination controller23, and a halogen lamp. The power supply and illumination controllerconverts AC voltage into the user-adjustable DC voltage supplied to thehalogen lamp. The lamp is located in a lamp holder that is attached tothe access door on the base plate. This positions the lamp forillumination of the specimen through the condenser assembly 57 andallows the lamp to be accessed for replacement. Heat from the lamp istransmitted through a heat shield and base plate and is dissipated tothe frame 3. The base plate provides structure for the illuminator 11and attaches to the bottom of the frame 3. In one embodiment, theilluminator operates at 90-264 VAC, single phase, 47-63 Hz, and has apower output of 35 W.

The illumination control panel 19 is located on the side of the lowerarm 47 of the frame 3 and provides an on/off switch 59 and control 23for adjusting illumination. The control panel 19 also holds thepotentiometers for adjusting the intensity of the white and green markindicator images.

FIGS. 8 and 9 illustrate one embodiment of the object marker subsystem18. Specifically, FIG. 8 is the assembled object marker subsystem 18, asit resides on the review station 2, and FIG. 9 is an exploded view ofthe object marker subsystem 18. The object marker subsystem 18 islocated on the review station 2 under the upper arm 27 and behind thestage 12. The marker 81 places physical marks at the locations ofelectronically marked target zones on a specimen mounted on the stage12. The marker's functions include, but are not limited to, physicallyplacing marks on the specimen to indicate sites where furtherinvestigation is suggested, physically marking the specimen to indicatethat the specimen has been reviewed, and providing for capping andsealing the marker 81 when not in use. The marker 81 is capable ofmarking on conventional glass slides, float glass slides, coatings onthe glass slide, glass coverslips, and plastic coverslips.

In one embodiment, the marker utilizes a Sharpie® pen, which can beobtained from the Sanford Corporation in Bellwood, Ill., to generate themark on the specimen. This pen is widely used and accepted for manuallymarking cover slips and slides. The solvent based ink marks reliably andquickly on glass or plastic cover slips, dries quickly so it does notsmudge easily, is fairly translucent, and is non-toxic. The markermotion is provided by a cantilevered arm 80 rotating about a fixed pivotpoint 83. This arrangement provides highly repeatable, reliable motionwith minimal frictional effects. One linear stepper motor 88 is used todrive the marker 81; however, other numbers and types of actuators arecontemplated. A sensor 87 is used to establish the home position of themarker 81.

The object marker subsystem 18 includes a capping/uncapping mechanism79. The uncapping motion is spring actuated; however, other types ofbiasing apparatus 90 are contemplated. The capping motion is actuated bythe stage 12. The stage 12 moves to a specific position to push themarker cap 84 into an engagement position with the marker 81. The stage12 includes a pin 97 or other type of protuberance to contact the caparm 82 and drive the cap arm 82 into the engagement position.

The marker 81 is positioned at a tilt angle of approximately 24°;however, other angles are contemplated. Orienting the marker 81 at anangle positions the marker 81 closer to the optical axis, whichminimizes the required stage travel and size. The marker 81 is heldfirmly during operation, without slippage or backlash due to variationin marker manufacturing. The marker holder 89 is located on the rightside of the frame upper arm 27. The marker 81 is easy to load andunload. The marker holder 89 weight is minimized to prevent anyincremental weight from contributing to potential marker impact issueson the specimen. In addition, the marker holder 89 can include aclamping mechanism to securely hold the marker 81 and is structurallyrobust enough to prevent damage due to accidental bumps.

The marker cap 84 provides a repeatable and reliable airtight sealcompliant with the manufacturing variation of the marker 81. The cap 84includes a replaceable diaphragm 85 to allow for replacement due to wearand damage. This diaphragm 85 is easily replaced by the user. Thediaphragm 85 has a combination of a low insertion/extraction force and ahigh seal compliance to assure reliability.

During marker 81 loading, the marker arm 80 moves to a home position (upin the current embodiment) and the marker cap 84 moves to an disengagedposition. The marker 81 is manually loaded and clamped in the holder 89.The RS 1 then proceeds to make a sequence of marks to calibrate themarker's vertical position relative to a focal position of the specimen.During marking, the marker arm 80 raises the marker 81 to the homeposition. As the marker arm 80 is raised, the spring loaded cap arm 82releases from the marker 81 and the cap 84 is biased away from themarker 81. In some embodiments, the stage 12 will position the specimeninto a marking position relative to the marker 81. Next, the marker arm80 lowers the marker onto the specimen with a controlled velocity, andthe stage 12 moves the specimen relative to the marker 81 to create themark. In some embodiments, the stage 12 makes multiple correspondingmoves to repeatedly mark the specimen in an over-writing fashion. Themark is a combination of line segments and/or arcs that at leastpartially bound the object of interest, an example of which is shown inFIG. 16. The marker arm 80 raises and maintains this marker position iffurther marks are required. If there are no more marks to be made, themarker arm 80 returns the marker 81 to the home position. The stage 12then moves the cap arm 82 and cap 84 into the engagement position. Themarker arm 80 lowers the marker 81 into the marker cap 84.

In one embodiment, the maximum allowable normal force (Z-direction) themarker 81 may transmit to the specimen is 30 g. This is intended tominimize the marker impact on the specimen. To prevent cell migrationdue to marker loading on the specimen, in particular, a coverslip may belocated over the specimen. A high impact may cause cell migration and/ormarker 81 bounce.

To ensure accuracy, the marker 81 should be calibrated each time it isreplaced. Calibration is recommended due to the relatively high accuracyneeded for the maker location and the corresponding large variation inthe manufacturing tolerances of the marker 81. A calibration procedureis used to establish the position of each new marker 81 to ensure that amark is placed accurately with respect to a marked target zone. Oncecalibrated, the dimensional variation between the mark indicator centerand the physical mark center during repeated cycles is preferably about±0.1 mm or less.

FIGS. 10-12 depict the mark indicator module 20. The module 20 islocated in the upper arm 27 of the review station frame 3, directlyabove the motorized nosepiece 14. The bottom of the mark indicatormodule 20 interfaces to the review station 2 via an optical mount ring.The mark indicator module 20 produces a shaped pattern 470 in the FOVthat indicates to the user whether a target zone is marked or not. Theshaped pattern 470 can be a line segment, an arc, or combinationsthereof, for example, an “L” or “V” shape. The mark indicator 20 is alsoa means for the user to locate material in the FOV prior to electronicmarking or a feature present in the optical path utilized inestablishing or verifying registration, i.e. a fixed reference withinthe FOV to which fiducial marks on the slide may be aligned by the user.In one embodiment, the mark indicator 20 is a device that produces anLED-illuminated pattern 470 that overlays the view of the specimen seenby the user. The pattern 470 is produced by directing light from an LED450 through a mask 452, focusing and combining the resulting pattern 470in the optical path 466 between the objective 43, 45 and the opticalinstrument 10. The mark indicator 20 includes an illumination lightsource 450 (white and green LEDs), a diffuser 454, a mask 452 with anopening the shape of the indicator pattern 470, an aperture 456, a lens458, a beam splitter 460, a housing 462, and pattern alignment andfocusing features. In one embodiment, the illumination light source 450,the diffuser 454, the mask 452, the aperture 456, the lens 458, focusingoptics 464, and the beam splitter 460 are in serial relationshipbeginning with the light source 450.

The illumination source 450 includes two green LEDs 451 and two whiteLEDs 451; however, any number and/or color of LEDs may be used and,depending on the particular application, illumination sources other thanLEDs may be used, such as a halogen bulb. When the green LEDs 451 areon, a green shaped pattern 470 is visible to the user looking into theeyepieces 29 of the review station 2. Similarly, when the white LEDs 451are on, a white shaped pattern 470 is visible to the user. The operationof the LEDs 451 is mutually exclusive, such that either of the sourcesof illumination (green or white) can be on, but not both. The LEDs 451are directed at the diffuser 454, which ensures that the pattern 470 isuniformly illuminated by the LEDs 451.

In the illustrated embodiment, the mask 452 is a chrome deposit on aglass substrate in the shape of the indicator 470, where the shape isclear and the background is opaque (chrome). This allows the lightgenerated by the LEDs 451 to pass through the mask 452, therebygenerating the shape 470 seen by the user. The aperture 456 reducesstray light and limits the numerical aperture of the system. The lens458 allows the pattern 470 to be properly focused in the optical path502. The beam splitter 460 combines the pattern image into the reviewstation optical path 466. The housing 462 offers mechanical support forthe optical and electrical elements of the mark indicator 20. Thepattern alignment and focusing features allow the pattern 470 to beproperly centered (translated), rotated, and focused so that the pattern470 appears at the correct orientation, position, and in focus in thereview station FOV. Electrically, the mark indicator LEDs 451 connect todigital output lines from a digital output port 453 to the processor 8.The brightness of the illumination source 450 can be adjusted by varyingseries resistance in the output lines.

Referring now to FIG. 16, an “L” shaped mark indicator pattern 470 isshown. The dimensions shown are approximate and for illustrativepurposes only. In the embodiment shown, the pattern 470 is about 1 mm byabout 1 mm and about 0.5 mm wide; however, the size and shape of thepattern 470 can be chosen to suit a particular application. As can beseen, the pattern 470 at least partially bounds the marked target zone472 on two sides.

The user interface 4 is illustrated in FIG. 6. The user interface 4 istypically located on a work surface adjacent to the review station 2.The user interface 4 is an input device intended for use when the user'svisual attention is required for viewing in the review station 2. Theuser interface is a two-piece plastic housing 50 with five input/outputdevices. The user interface 4, however, may include any number and/orcombination of input/output devices to suit a particular application. Inone embodiment, the input/output devices include a specimen positioninput device (“SPID”) 52 and four independent buttons 54 facilitatingthe following commands: next, previous, objective, and mark. The userinterface 4 allows the user to manually position the stage 12 via theSPID 52. The SPID 52 is used for centering FOIs within a target zone forsubsequent review and marking by a user.

The housing size and shape is intended to provide ergonomic benefit tothe user as well as the physical space required for the electroniccomponents. The size is preferably small to minimize the space requiredfor the user interface 4. The housing 50 may be fabricated from sheetmetal, plastic, machined components, or combinations thereof. Thehousing base 51 may include non-skid rubber feet attached to the bottomto prevent slipping during use. The rubber feet are adequately spaced toprevent tipping during button actuation. In one embodiment, the housing50 is shaped to orient the input/output devices at about 15° relative tothe work surface to provide for ease of use.

In one embodiment, the SPID 52 is a button joystick that utilizeshall-effect sensor technology to sense displacement along two orthogonalaxes. The SPID 52 utilizes the magnitude of the displacement along thetwo axes to control the speed and direction of the stage 12. The SPID 52outputs are two analog signals ranging from 0 to 5 volts. The otherinput devices are push-buttons 54, but could be any similar device, suchas a toggle switch. The buttons are positioned relatively close to theSPID 52 for ease of use. The button force and travel characteristics aresimilar to a computer “mouse” feel with relatively low actuation force(e.g., <200 g), and high tactile response for user feedback. The userinterface 4 houses an electrical interface 53 that is in datacommunication with the RS processor 8 via a cable 99.

The following is one example of an application of the user interface.The SPID 52 can be used to align fiducial marks with a mark indicator470 to establish registration. Two of the buttons 54 function as nextand previous commands for sequencing through a predetermined number ofFOIs during an autolocate mode 610 and through a series ofelectronically marked target zones (if any) during a review mode 612.The buttons 54 trigger the transition to the next or previous FOIs in auser-triggered autoscan. A third button 54 can be used to electronicallymark or unmark a target zone. A fourth button 54 can be used to togglebetween multiple objective lenses, for example 10× and 40×.

The console 6 is illustrated in FIG. 7. The console 6 is also typicallylocated on the work surface adjacent to the review station 2. Theconsole 6 is an input/output interface for the review station 2 intendedfor use when the user's visual attention is not required for viewing inthe review station 2. The console 6 includes a housing 30, a keypad 32,a display 36, a serial interface/controller, and a cord 39. The housing30 protects and supports the electronic components contained within andmay be fabricated from sheet metal, plastic, machined components, orcombinations thereof. The housing 30 size and shape is intended toprovide ergonomic benefit to the user as well as the physical spacerequired for the electronic components. The width of the housing baseshould be optimized to provide adequate button spacing and to minimizetipping during keypad entry and to minimize overall console profile. Thehousing base may include non-skid rubber feet attached to the bottom toprevent slipping during use. The rubber feet are adequately spaced toprevent tipping during button actuation. In one embodiment, the consoleis about 3.6″ high, about 6.3″ deep, and about 4.6″ wide.

In one embodiment, the display 36 is a 128×64 backlit, dot-matrixgraphics LCD. The character height is about 6 mm. The character heightshould be optimized to provide adequate viewing from a nominal distanceof about 3 feet; however, the text height could be varied for anyparticular application. The display 36 has a viewing angle of about 35°to provide for optimal viewing. The display 36 is viewed through aprotective window 31 on the console bezel 33. The display 36 presentsthe user with the following information: system status includingprogress updates, display error log, maintenance record, usage historyentries, prompts and error messages, user preference menus, diagnosticinformation, and the current operational mode of the device; however,this list is not exhaustive.

The keypad 32 may be a membrane switch with a graphic overlay. Themembrane switch utilizes a metal dome (in a specific “dummy dome”construction method) to enhance switch tactile feedback. In one example,the keypad actuation force is less than 250 g. The keypad 32 has thefollowing keys: 0-9 (numeric), soft (software definable) keys, andcancel. The soft keys 34 are buttons in the vicinity of the display 36.The labels associated with these keys 34 appear on the display 36. Thelabels and the actions associated with pressing the soft keys 34 maychange depending upon the current operational mode. Keypad 32orientation is preferably 15° to provide for ease of keypad entry.Examples of console functions performed via the keypad 32 includeaccepting inputs from the user, inputting user identification duringlogin, manual slide ID input, changing the operational mode (autolocate610, review 612, autoscan 614, 622, and manual review 636) of the RS 1,accept user input to complete specimen review, accept user input tologout, accept input of user preferences, and accept user input tocancel a slide review, among others. The serial interface/controller isa PCA that has an RS-232 interface to a Parallel-Serial Interface Module(PASEIM) on the processor 8. The serial interface/controller controlsthe LCD, stores fonts, graphics, and macros, and scans and encodes thekeypad 32 and operator interface buttons 35.

FIG. 14 depicts one embodiment of a target zone mark 474, the dimensionsof which are approximate and for illustrative purposes only. The mark474 is essentially an “L” shape; however, the size and shape of the mark474 can vary to suit a particular purpose. For example, the mark caninclude line segments, arcs, or combination thereof, which at leastpartially bound a target zone 472.

An embodiment of the slide reviewed mark 480 is shown in FIGS. 15A and15B. The mark 480 is a vertical line about 1 mm thick and about 5 mm inheight centered on a screen-printed left hand arc of the specimen slideshown in FIG. 15A. FIG. 15B is a partial enlarged view of FIG. 15A andshows dimensions, which are approximate and illustrative only, for oneembodiment of a slide reviewed mark 480. The size and shape of the mark480 can be selected from line segments, arcs, or combinations thereof.

FIG. 17 is a graphical representation of one possible electricalarrangement for an embodiment of a RS 1 in accordance with theinvention.

FIG. 18A depicts one embodiment of an accelerometer 490 that can be usedwith an imaging system for accommodating vibration errors that occurduring imaging. The dimensions shown are approximate and forillustrative purposes only. The size and model accelerometer 490 usedwill vary to suit a particular application. The accelerometer 490 istypically a high-precision, high-resolution device utilizing a ceramicshear mode sensing element. The accelerometer 490 shown has a voltagesensitivity of 100 mV/g, a measurement range of +/−50 g [+/−491 ms2peak] for +/−5V, a frequency range of 0.2 to 20,000 Hz (preferably 1 to10,000 Hz), and an excitation voltage of 18-30 VDC/2-20 mA. Theaccelerometer 490 can be a commercially available product, such as model352C66 available from PCB Piezotronics of Depew, N.Y. The accelerometer490 includes an electrical connector 494 for electrical communication toa processor.

FIG. 18B depicts the accelerometer 490 of FIG. 18A mounted on oneembodiment of a frame 492 for the imaging system. Mounting is via athreaded end 496 on the accelerometer 490; however, the mounting can bedone via any known methods of fastening. The accelerometer measures thevibration the imaging system experiences while imaging a specimen. Thevibration measurement is taken for each portion of the specimen that isimaged. If the measurement exceeds a set threshold, the system willre-image the portion of the specimen that was being imaged immediatelyfollowing the vibration measurement. If the number of portions re-imagedexceeds a set threshold, the system may reject the specimen.Alternatively, if the vibration measurement exceeds a second setthreshold, the system may reject the slide without re-imaging thespecimen portion.

Referring to FIG. 19, one embodiment of the slide holder assembly 100 isshown. A first platform 104 of the slide holder assembly 100 is movablyconnected to a base 102. The first platform 104 slides relative to thebase 102 via a series of rails 130 on the base 102 that contact matingsurfaces 128 on the underside of the first platform 104. This movablerelationship of the first platform 104 relative to the base 102 may beachieved by any suitable mechanism including, but not limited to, atongue and groove mechanism, a sliding dove-tail joint, a rollerassembly, a ball bearing assembly, or combination thereof. A secondplatform 106 is attached to the first platform 104. Attachment of thesecond platform 106 to the first platform 104 can be by welding,bonding, rivets, bolts, machine screws, or other suitable attachmentmethods. The base 102 can be connected to an actuating table 108. Theconnection between the base 102 and the actuating table 108 may be apermanent attachment or a temporary attachment, so that the base 102 maybe detached from the actuating table 108 and a different embodiment of aslide holder assembly 100 may then be configured. The actuating table108 itself may be fixed to ground or temporarily attached to a suitablesupport via a lock and release mechanism.

Respective upper surfaces of the base 102, the first platform 104, thesecond platform 106, and the actuating table 108 are shown as beingsubstantially rectangular in shape; however, the shape of these surfacesmay be circular, triangular, oval, elliptical, square, an irregularcontour, or any other suitable geometric shape. In addition, the base102, the first platform 104, the second platform 106, and the actuatingtable 108 may be any suitable three dimensional solids and are notlimited to machined or molded rectangular solids.

The base 102, the first platform 104, the second platform 106, and theactuating table 108 may be made from the same or differing materialsthrough molding, machining, casting, sculpting, cutting, etching,thermosetting, or other suitable fabrication process. The class ofsuitable materials for the base 102, the first platform 104, the secondplatform 106 and the actuating table 108 includes metals, alloys, woods,plastics, composite materials, resins, or any other sufficiently strongand durable material. Similarly, any component part of the holderassembly 100 may be made of any material as may be suitable to theoperational requirements of the component part. In addition, any of thecomponent parts disclosed in this specification and the variousembodiments of the invention illustrated herein, may be hollow, solid,or a combination of both, as required for a particular application.

A first slide positioning member 110 and a second slide positioningmember 112 are shown connected to the second platform 106. A stop 113,in this embodiment of the invention, is an edge wall of a recessedcavity within which the slide 116 may be positioned by the first slidepositioning member 110 and the second 112 slide positioning member. Thestop 113 may also be, for example a raised pin, a tab, a recessedcavity, a ridge, a groove, any combination of the preceding structures,or any other suitable slide restraining structure. In variousembodiments of the invention, the slide 116 is preferably manufacturedof float glass, which is extremely flat. The open configuration of thesecond platform 106 in this embodiment of the invention allows viewing,imaging, and marking systems easy access, in conjunction with roboticsystems for slide positioning and transfer to and from the imagingsystem.

The slide actuating mechanism 114 in this embodiment is manufacturedfrom formed sheet metal, although other suitable materials may be used.The first slide positioning member 110 and the second slide positioningmember 112 engage the actuating mechanism 114 when the first platform104 is moved relative to the base 102. This occurs, in part, because theactuating mechanism 114 is attached to the base 102. The actuatingmechanism may be bolted, welded, chemically bonded, or otherwisephysically connected to the base 102. The actuating mechanism 114engages the first slide positioning member 110 and the second 112 slidepositioning member at protrusions 134 extending from the actuatingmechanism 114. Alternative actuating mechanisms include, a cam system,an actuating motor, an internal spring system, or other suitableapparatus or structure.

Still referring to FIG. 19, the biasing of the first 112 slidepositioning member 110 and the second 112 slide positioning memberstowards the slide receiving area with a slide 116 in contact with thestop 113 is achieved by the presence of a first resilient member 120 anda second resilient member 118 in contact with the respective positioningmembers 110, 112. The slide positioning members 110, 112 are rotatablymounted to the second platform 106. The resilient members 120, 118 areconnected to the positioning members 110, 112 so that when the resilientmembers contract, they cause the slide positioning members 110, 112 torotate towards the slide receiving area. When the resilient members 120,118 are springs, they may be connected to the slide positioning members110, 112 and to the second platform 106 through drilled holes 132 in themembers 110, 112, and the second platform 106 respectively. Theresilient members 120, 118 and the positioning members 112, 110 may alsobe connected via chemical bonding, screws, bolts, catches, hooks, orother suitable means. The resilient members 120, 118 may be springs,elastic bands, collapsible shocks, or other suitable devices in variousembodiments of the invention.

The resilient members 120, 118 bias the first 110 slide positioningmember and the second 112 slide positioning member towards the slidereceiving area. This biasing is opposed when the actuating mechanism 114causes the slide positioning members 110, 112 to pivot away from theslide receiving area to facilitate removal of a slide 116 from theassembly 100. The actuating mechanism 114 in this embodiment of theinvention has two raised protrusions 134. When the first platform 104slides relative to the base in a first direction, the actuatingmechanism 114, which is fixed to the base 102 makes contact with eachslide positioning member 110, 112, such that one protrusion 134 contactsone member. The protrusions 134 move against the slide positioningmembers 110, 112 as the base 102 and first platform 104 continue to moverelative to one another, in a first direction, which causes the members110 and 112 to rotate away from the slide. When the base 102 and thefirst platform 104 move in the opposite direction, the protrusions 134disengage from the positioning members 110, 112 and the tension suppliedby the resilient members 120, 118 causes the respective slidepositioning members 110, 112 to move toward and contact the slide 116,which moves the slide 116 into contact with the stop 113. The slide 116is held in place by opposing contact forces originating from the stop113 and the slide positioning members 110, 112 as biased by theresilient members 120, 118.

Referring to FIG. 20, another embodiment of the slide holder assembly100 is shown. In this view of the holder 100, only one slide positioningmember 112 is visible. This embodiment is similar to that describedherein above with respect to FIG. 19. For example, the base 102, thefirst platform 104, and the second platform 106 are very similar tothose shown in FIG. 19. The embodiment shown in FIG. 20 does differ fromthat shown in FIG. 19 in some respects. For example, this embodimentdepicts a motor 122 for driving the actuating table 108. The motor 122may be a solenoid, servo, or any other suitable actuating device. Inaddition, this embodiment shows the actuating mechanism 114 as a solidmachined member. The protrusions 134 extending from the solid memberactuating arm 114 in FIG. 20 are raised round cylinders. This differsfrom the protrusions 134 in FIG. 19, which are shown as cut metal tabs.

Referring to FIG. 21, the arrow 124 illustrates the direction in whichthe actuating table 108 may move the base 102, the first platform 104,and the second platform 106. This view of the holder 100 shows a slide116 in an unsecured position in the slide receiving area of the secondplatform 106 with the first slide positioning member 110 and the secondslide positioning member 112 actuated away from the slide 116 by theprotrusions 134 of the actuating mechanism 114. Arrow 126 illustratesthe motion of the first platform 104 relative to base 102. The resilientmembers 120, 118 are connected to the second platform 106 and areoriented substantially parallel with the motion of the platform. Arrow126 shows one possible axis of motion of the base 102 relative to thefirst platform 104. The resilient members 118, 120 exert a force on thefirst positioning member 110 and the second positioning member 112,respectively, to engage the slide 116. When the first platform 104moves, the slide positioning members 110, 112 contact parts of theactuating mechanism 114, such as the protrusions 134. The contactbetween the slide positioning members 110, 112 and the actuatingmechanism 114 creates a force which opposes the biasing of thepositioning members 110, 112 by the resilient members 120, 118 towardsthe stops 113 of the slide receiving area. The biasing opposing force ofthe actuating mechanism 114 may be adjusted by the movement of the firstplatform 104 so that the first and second slide positioning members 110,112 come into secure contact with the slide 116, thereby moving it intosecure contact with the stop 113. The stop 113 is defined as any slideposition restricting element. In some embodiments of the invention, aslide restraining system such as a series of vacuum channels, may beincorporated into the assembly 100 to exert a normal force on the slide116 to hold the slide 116 firmly in place on the second platform 106once against the stops 113.

FIG. 22 shows the same embodiment of the invention as shown in FIG. 21,but with the actuating mechanism 114 in a different position as a resultof the first platform 104 being moved with respect to the base 102. Themotion of the first platform 104 and the disengagement of the actuatingmechanism 114 from the slide positioning members 110,112 causes theslide positioning members 110,112 to contact the slide 116 and secure itin a position abutting the stops 113. The slide positioning members 110,112 are shown in a slide contacting position. The slide 116 is securedbetween the members 110, 112 and the stops 113.

FIG. 23 depicts a perspective view of the slide holder assembly 100,wherein the actuating mechanism 114 is restraining a first slidepositioning member 110 and a second slide positioning member 112 in anopen state.

Referring to FIG. 24, some of the constituent parts of some embodimentsof the invention are shown. The second platform 106 is shown as amachined or cast metal frame with pre-drilled holes in this embodimentof the invention. The first slide positioning member 110 and the secondslide positioning member 112 are shown in position to be rotatablymounted to the second platform 106. A first resilient member 120 and asecond resilient member 118 are in position to be connected to theirrespective slide positioning members 110,112. The resilient members 120,118 are metal springs in this embodiment of the invention. A raised stop113 is fabricated as part of the metal frame forming the second platform106.

Referring to FIG. 25, a close up the slide receiving area isillustrated, wherein the slide 116 is shown in a position secured by afirst slide position member 110, a second slide positioning member 112,and various stops 113. The first slide positioning member 110 is shownin contact with the slide 116 at a contact region at an end of the slide116. Similarly, the second slide positioning member 112 is shown incontact with the slide 116 at another contact region along an edge ofthe slide 116. When the slide 116 is in this position, the actuatingtable 108 may be used to move the slide 116, as desired, under imagingoptics.

Referring to FIG. 26A one embodiment of the first slide positioningmember 110 is shown. Similarly, referring to FIG. 26B, one embodiment ofthe second slide positioning member 112 is illustrated. Theseembodiments of the slide positioning members 110, 112 are depicted inFIG. 24 prior to their assembly to the second platform 106. The slidepositioning members 110, 112 each have a slide contacting end 140, anactuating mechanism contacting region 142, a resilient member attachmentpoint 144, and pivot region 146 for mounting to the second platform 106.The slide holding members 110, 112 may take on any geometric shape thatallows them to be mounted to the second platform 106, while retainingthe ability to move, make contact with a slide 116, and be operativelyengaged and disengaged from the slide 116 by an actuating mechanism 114.The configuration of the slide positioning members 110, 112 and theprotrusions 134 can be designed so that both members 110, 112 actuatesimultaneously or serially, as desired. In various embodiments slides116 designed for use with cytological specimens may be used. Slides 116that contain cytological specimens stained with thionin-phenolsolutions, including stains with phenol derivatives are well suited foruse in the assembly 100.

Referring to FIG. 27, one embodiment of a slide 116 for use in theinvention is shown. A float glass slide 116 is depicted with variousdimensions and tolerances in inches for its fabrication. Float glass ismade by forming a layer of molten glass on a molten metal such as tinand subsequently cooling the molten glass to solidification. Because ofthe method of manufacture, the glass surface is extremely flat,obviating the surface irregularities and waviness inherent in producingglass with rollers. Other types of slides 116 may also be used invarious embodiments of the invention.

FIG. 28 includes a schematic representation of a generally circular cellspot 202 on a slide 200 to be imaged. The cell spot 202 is the region ofinterest of this slide, on which a cytological specimen lies, forinstance. The cell spot 202 may be roughly two centimeters in diameter.In one embodiment of the invention, it is desired to acquire imagescovering substantially all of the cell spot 202 and which deviate froman optimal focus position no greater than about ±4 μm, as measured byposition of an optical component along the focus axis generally normalto the plane of the slide 200. In this embodiment it is further desiredto complete the imaging of the cell spot 202 of the slide 200 quickly,for example within 165 seconds or less.

An embodiment of the invention utilizes three fiducial marks 204, 206,208 depicted in FIG. 28 located at predetermined coordinate locations onthe slide 200. In this embodiment, the fiducial marks 204, 206, 208 havea known shape with a rapid transition from dark to light when in focus.By identifying and recording the optical component focus axis locationat which a sharp edge transition occurs, the global focal surface (inthis case, a plane) containing the sample can be estimated. Thisestimated global focal plane serves as a guide across the complete cellspot and helps to establish a boundary condition that the focus axisshould not extend beyond under normal operating conditions.

FIG. 29 is a schematic representation of a global focal surfacedetermination flowchart 220 summarizing certain process steps inaccordance with one embodiment of the present invention. In thisembodiment, a system is contemplated which includes a two-axis slideimaging stage. The two orthogonal axes are the index and scan axes 210shown in FIG. 28. At a given time, the system may perform either acoarse positioning move to get close to a desired location, or a scanmovement. In this embodiment, a scan pass from one edge of the cell spot202 to an opposite edge includes of a series of short moves, eachapproximately 370 μm long. Motion may be provided by a Piezo CeramicLinear Motor (PCLM) packaged by the Anorad Corporation under the name ofPico Stage. Also, a linear encoder reports the actual position of thestage. The stage may be guided along its axis using crossed rollerbearings. An optical sensor may be provided to indicate home position ofthe stage. The scan axis supports the slide holder and mounts normal tothe index axis, in this embodiment. Naturally, other motion systemscould be employed and will be apparent to those skilled in the art.

FIG. 29 shows that according to one embodiment, the first step 222 indetermining the global focal surface is to determine the index axis andscan axis coordinates of the first fiducial mark, which is shown as FM#1204 in FIG. 28. In this embodiment, the centroid of FM#1 204 isdetermined to within ±10 μm of the stage home position for the firstfiducial mark FM#1 204. In step 224 of FIG. 29, the focus axiscoordinate corresponding to FM#1 204 is determined. This is done byperforming a coarse focus action at a position corresponding to FM#1204. In this embodiment, a coarse focus action is performed by imagingFM#1 204 at an initial physical position, here, a z-position, whichcorresponds to a coordinate on the focus axis, and by determining afocus score corresponding to that coordinate. One method of determininga focus score, developed by Brenner et al. (“An Automated Microscope forCytological Research: A Preliminary Evaluation,” Brenner et al., TheJournal of Histochemistry and Cytochemistry, Vol. 24, No. 1, pp.100-111, 1976, the disclosure of which is incorporated herein byreference in its entirety), is to calculate the average change in graylevel between pairs of points separated by n pixels, as follows:

$\begin{matrix}{{f(z)} = {\sum\limits_{j}\;{\sum\limits_{i}\;\left\{ {{G_{i}(z)} - {G_{i + n}(z)}} \right\}^{2}}}} & (1)\end{matrix}$where the index (i) ranges over all image points, in order, along a scanline (j); n is a small integer, especially 2; z is the focus axisposition; and G_(i) is the transmission gray level for point i. It isthen desired to obtain a scan axis coordinate which provides a maximumvalue of focus score, f(z).

FIG. 30 is a schematic representation of a sample plot of focus axisposition versus focus score in accordance with one embodiment of thepresent invention. The plot 260 of FIG. 30 shows values of focus score,f(z), at positions corresponding to various coordinates on the z-axis(focus axis). One embodiment of the invention applies an algorithm tofacilitate the determination of a z-axis (focus axis) coordinate towithin ±4 μm of the coordinate providing the maximum focus score. Inthis embodiment, the imaging optics include an objective lens (numericalaperture of 0.25, magnification of 10×), a relay or tube lens(magnification of 1.8×), and a camera. The camera is used to captureimages of discrete portions of the slide, and has the followingfeatures: an 8-bit resolution (providing 256 gray-scale values);progressive scan (non-interlaced) analog video signal to reduce theeffect of any movement or vibration during image capture; asynchronoustrigger reset mode, to allow for synchronized image capture; 25 Hz framerate (minimum) for maximum throughput; square 8.3 μm pixels, square toeliminate x-y scaling and an 8.3 μm size to reduce magnificationrequirements thereby increasing the depth of field; 782×582 pixel arraysize, the minimum array size for the desired resolution; a sensitivityof 400 Lux, F4 for imaging; external electronically controlled shutter;a standard “C” mount for attachment; and a horizontal frequency of about15,625 kHz, providing for a pixel clock rate of about 12 MHz. The use ofa Sony model SC-8500CE (Sony Electronics Inc., USA) camera is consistentwith these specifications.

The algorithm used in this embodiment during the coarse focus procedureapplied at the first fiducial mark FM#1 204 is as follows, and isdepicted in the plot 260 of FIG. 30. A picture is taken at an initialz-position (z=0) and its focus score, f(z) of Equation (1), iscalculated. This is the first point, and the resulting focus score isdepicted at box 1 of FIG. 30 (the boxed points are labeled in FIG. 30according to the order in which they are measured). This score is set asthe temporary maximum score. The position of the focus axis is thenmoved 64 μm below the initial z-position, relative to the surface of theslide. Note that this may be done either by moving an element of theoptical system or by moving the slide, although moving an element of theoptical system is generally easier. The position is imaged, and a focusscore is determined at that position, depicted at box 2 of FIG. 30. Thesecond focus score is compared to the first focus score. If the secondfocus score is less than the first focus score, then the focus axis ismoved to a position 64 μm above the initial z-position. If the secondfocus score is greater than the first focus score, then the temporarymaximum score is reset as the second focus score. The focus axis ismoved in 64 μm increments until an image is captured at a z-positionwith a lower focus score than the current maximum focus score. In FIG.30, the z-position corresponding to the point depicted at box 4 is justsuch a z-position. The idea is to continue making relatively large movesuntil an image has been captured on either side of a peak. Once the peakhas been narrowed down to a 64 μm range, the current step size isdivided by two. The focus axis is moved to the z-position substantiallyequidistant from the two positions on either side of the peak. A newimage is taken and focus score calculated. If the score is greater thanthe maximum, the step size is divided by two again, and the nextz-position is set to the current z-position plus the new step size. Theprocess is continued until the step size reaches either 4 μm, 2 μm, orother predetermined small value, as shown in FIG. 30, and the image withthe highest focus score is returned, which would be the imagecorresponding to the fifth z-position in the plot 260 of FIG. 30.

Thus, a value of focus axis coordinate is obtained for fiducial markFM#1 204, thereby completing step 224 of FIG. 29. The next step 226 indetermining the global focal plane in this embodiment is to determinethe index axis and scan axis coordinates of the second fiducial mark,FM#2 206, shown in FIG. 28. In this embodiment, the centroid of FM#2 206is determined to within ±10 μm of the stage home position for the secondfiducial mark FM#2 206. After steps 222 and 226, where index axis andscan axis coordinates are determined, the embodiment will generate aslide error and flag the slide electronically or physically if thedistance between FM#1 204 and FM#2 206 is greater than 500 μm. Afterstep 226, the embodiment includes the performance of another coursefocus procedure, as discussed above, to complete step 228, thedetermination of a focus axis coordinate of FM#2 206. Then, the x-yposition (index axis and scan axis coordinates) of the third fiducialmark, FM#3 208 of FIG. 28, is estimated based on the positions of FM#1204 and FM#2 206. The stage is then moved to capture an image at thiscalculated position. If the third fiducial mark, FM#3 208, is greaterthan 300 μm from the computed location, the search results are rejected.Also, the slide is flagged and rejected if a fiducial mark cannot belocated. Assuming the third fiducial mark is found, a coarse focusaction is performed to determine the focus axis coordinate of FM#3 208,completing step 234 of FIG. 29. Step 236 is the determination of anindex axis slope based on the coordinates for FM#1 204 and FM#2 206,which may be performed by computing the following quotient:index axis slope=(z ₂ −z ₁)/(x ₂ −x ₁)  (2)where z₂ is the focus axis coordinate of FM#2 206, z₁ is the focus axiscoordinate of FM#1 204, x₂ is the index axis coordinate of FM#2 206, andx₁ is the index axis coordinate of FM#1 204. In this embodiment, theindex axis slope is checked against a threshold value, for example, 2 μmfocal axis distance over 25 mm along the index axis. If the index axisslope is greater than the threshold value, the slide is flagged anddenoted physically or electronically as having an error. Step 238 ofFIG. 29 is similar to step 236. Here, the scan axis slope is determinedby calculating the following quotient:scan axis slope=(z ₃ −z ₂)/(y ₃ −y ₂)  (3)where z₃ is the focus axis coordinate of FM#3 208, z₂ is the focus axiscoordinate of FM#2 206, y₃ is the scan axis coordinate of FM#3 208, andy₂ is the scan axis coordinate of FM#2. In this embodiment, the scanaxis slope is checked against a threshold value, for example 2 μm focalaxis distance over 25 mm along the scan axis (although this value doesnot necessarily have to be set equal to the threshold value for theindex axis slope). If the scan axis slope is greater than the thresholdvalue, the slide is flagged and denoted physically or electronically ashaving an error. A focus intersect is calculated in step 240 to completethe characterization of the global focal plane in this embodiment asfollows:focus intersect=z ₁−(index axis slope)*x ₁−(scan axis slope)*y ₁  (4)where x₁, y₁, and z₁ are the index axis, scan axis, and focus axiscoordinates, respectively, at the point corresponding to the position offiducial mark FM#1 204; and the index axis slope and scan axis slope areas determined in Equations (2) and (3), respectively.

Another embodiment of the invention involves the performance of a scanpass over the area of interest of a slide. FIG. 31 is a schematicrepresentation of a scan pass flowchart 270 summarizing certain processsteps in accordance with one embodiment of the present invention. Instep 272 of FIG. 31, a coarse focus action, as discussed above, isperformed at a first position on the area of interest of the slide. Inone embodiment, this first position corresponds to the estimated centerof the cell spot 202 on the slide. The scan pass starts at the center ofthe cell spot, and moves radially outwardly. This maximizes coverage ofthe cell spot and enhances efficiency of the scan process. Theembodiment takes advantage of the implementation of an initial coarsefocus followed by periodic fine focus actions during the scan pass. Inone embodiment, a coarse focus begins by moving the focus axis relativeto the surface of the slide to a position corresponding to a focus axiscoordinate and incrementally raising and lowering the stage, searchingfor the location at which the focus score of the image is at a maximum.Unlike coarse focus actions, fine focus actions begin with theassumption that the current location is close to an in-focus location.Consequently, the search pattern for fine focus actions is moreefficient.

Step 274 of FIG. 31 is the determination of a fine focus jurisdictionfor the first point. The concept of a fine focus jurisdiction of thisembodiment allows more efficient performance of the scan pass, such thatfocus actions do not have to be performed at each location at which animage is acquired. Instead, a focus action (coarse or fine) has a“jurisdiction” of influence, wherein any other image can reasonably relyupon that previous focus action for guidance on the focal plane. Notethat FIG. 31 uses the term “fine focus jurisdiction,” whether the focusaction taken at a given point is coarse or fine. A focus actionjurisdiction in this embodiment is defined as having a range of ±6images along the scan axis and ±3 images along the index axis. Anelliptical jurisdiction pattern applies for images that are not exactlyalong the scan or index axes from a previous fine focus action. If a newimage is not within any focus action jurisdiction, then a new focusaction occurs.

FIG. 32 is a schematic representation of an area of fine focusjurisdiction in accordance with one embodiment of the present invention.In this embodiment, the focus action jurisdiction 310 is generallyelliptical in shape and has associated with it a predetermined lengthX_(coverage) 312 along the minor axis of the ellipse and a predeterminedlength Y_(coverage) 314 along the major axis of the ellipse. Also, inthis embodiment the major axis of the ellipse is parallel to the scanaxis, and the minor axis of the ellipse is parallel to the index axis.

An embodiment of the invention begins with a fine focus action todetermine the focus score of the image at the best guess of the in-focusz-coordinate, determined using the representation of the global focalsurface, as in FIG. 29. In this embodiment, from the best-guesslocation, up to four more images may be taken and scored. Theserepresent images that are taken at +4, +8, −4, and −8 μm from the bestguess location. The image with the highest corresponding score isdeclared the in-focus image.

Step 276 of FIG. 31 is the imaging of a region about the first point. Atypical size of the area imaged during a single exposure in thisembodiment is 640×480 pixels, but may vary according to the camera used.There are approximately 2500 frames over the cell spot depicted in FIG.28.

In step 278 of FIG. 31, a scan pass move is made along the scan axis toa new position. In step 280, it is determined whether the new positioncorresponds to a point which is within at least onepreviously-determined fine focus jurisdiction. If not, a new fine focusaction is performed to determine an appropriate focus axis coordinate atthe new position, as shown in step 282, followed by the determination ofa fine focus jurisdiction surrounding the new position, shown in step284. In one embodiment, step 282 is performed using an initial estimateof an appropriate focus axis coordinate based on the determination ofthe global focal surface as shown in FIG. 29.

If the new position corresponds to a point which is determined to bewithin at least one previously-determined fine focus jurisdiction, step286 determines whether the point is within exactly one fine focusjurisdiction. If so, step 290 prescribes the use of the correspondingfocus axis coordinate correlated with the one fine focus jurisdiction,corrected for tilt. The tilt correction in this embodiment is based onthe global focal surface equation for the slide determined as shown inFIG. 29, and is a modification of the value of the focus axis coordinatecorresponding to the new position in an amount equal to the change inpredicted focal plane from the previously imaged position to the newposition.

If the new position corresponds to a point which is within more than onefine focus jurisdiction, then the determination of step 286 is negative,and step 288 of FIG. 31 prescribes the use of a weighted average of thecorresponding focus axis coordinate values, corrected for tilt asdiscussed above. This step involves the use of an inverse distanceweighting function to determine the in-focus value at the new point,such that the focus axis coordinate corresponding to the jurisdictionwhose center is closer/closest to the new point is more heavily weightedthan (any of) the focus axis coordinate(s) corresponding to the otherjurisdiction(s) in which the new point lies.

After an appropriate focus axis coordinate is determined for the newpoint, a region about the point is imaged in this embodiment, as shownin step 292 of FIG. 31. Then, in step 294, it is determined whethersubstantially all of an area of interest—here, a cell spot—has beenimaged. If so, the scan pass for this slide is complete, step 298. Ifnot, step 296 prescribes a move along the scan axis to a new point. Step300 determines whether this point is within the cell spot, and if not,step 302 calls for the repositioning to a new index axis coordinatewithin the cell spot. In this embodiment, the cell spot is consideredmade up of a center area and an edge area. For points inside the edgearea, in this embodiment, an arc detect is used so that arcs indicatingthe edge of a cell spot are not used in any analysis, and frames thatare believed to contain an arc are still imaged and may be analyzed.

In this embodiment, the scan pass process continues as shown in FIG. 31,with a determination made at each successive point of whether the pointlies within a previously-determined fine focus jurisdiction, followed bythe appropriate use of retrieved focus axis coordinates or theperformance of fine focus actions at that point.

The pattern shown in the cell spot 202 of FIG. 28 represents the stepsof the scan pass procedure of FIG. 31 completed over the entire cellspot. In this embodiment, an initial coarse focus action is taken at thecenter of the cell spot and the corresponding region is imaged. Since afocus action was taken, an area of fine focus jurisdiction is determinedabout this point, and the value of the focus axis coordinatecorresponding to this area is associated with points within this area.Then, the stage is moved to a position about 370 μm below the center ofthe cell spot, along the scan axis. It is determined that this pointlies within the fine focus jurisdiction of the first point, so no finefocus action is taken here. The focus axis coordinate of the first pointis used to image the second point, corrected for tilt. Moves continuealong the scan axis until a point is reached outside the area ofjurisdiction of the initial point. Then, a fine focus action isperformed at that point, and a new area of jurisdiction is determined.Moves continue until the edge of the cell spot is reached. A move is nowmade along the index axis to a new index axis coordinate, and movescontinue along the scan index in an direction opposite that taken in thefirst line of moves along the scan axis. The process continues until onehalf of the cell spot is imaged, and the process continues again fromthe center extending in the opposite direction along the scan axis tothat taken in the first line of moves from the center. The processcontinues until the entirety of the cell spot is imaged.

In one embodiment, to expedite identification of focus axis coordinatescorrelated with fine focus jurisdictions applicable at a specificlocation, the cell spot area (area of interest of the slide) ispartitioned into bins. The bins represent rectangular regions of thecell spot area. Every fine focus jurisdiction that intersects a bin isput into the bin. Thus, a search for focus axis coordinates correlatedwith fine focus jurisdictions applicable at a specific location can belimited to those coordinates that are assigned to the bin wherein thespecific location resides.

An embodiment of the invention includes numerous checks to ensure thatthe focus axis is tracking well to the in-focus plane. These include theestablishment of absolute maximum and minimum focus axis values that arebeyond the reasonable limit for an in-focus image; application of amaximum threshold to the value of the slope of the global focal surfaceas predicted by the fiducial mark locations; establishment of anout-of-bounds test for focus actions based upon the global focal surfaceas predicted by the fiducial mark locations; application of fiducialmark verification tolerances that demand that the location of thefiducial mark at the end of cell spot processing be within a particulardistance from the initial search at the beginning of cell spotprocessing; establishment of a minimal focus score for both coarse andfine focus actions such that images scoring below this threshold are notjudged as in-focus; establishment of a minimal number of fine focusactions that must occur across a cell spot; and establishment of amaximum percentage of fine focus actions that can occur at the extremesof the fine focus search region (±8 μm). Additionally, the embodimentmay include the use of a checksum variable to account for each movementalong the focus axis, in order to mitigate against data corruptionassociated with fine focus actions and cell spot partitioning.

In a further embodiment, after the acquired slide images have beenprocessed, the data is reduced to a collection of Objects of Interest,which may merit further analysis by a cytotechnologist. The objects ofinterest are first ranked to determine the top 100 most importantobjects of interest. Once the top 100 objects of interest are sorted byimportance, the objects of interest are mapped to slide coordinates.Once the mapping occurs, FOIs are computed. The FOIs are then organizedto present an efficient path, using the Traveling Salesman (Hungarian)algorithm, for review by the cytotechnologist at the reviewing station.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. For example, theinvention may be embodied in methods pertaining to electron microscopefocusing systems. The foregoing embodiments are therefore to beconsidered in all respects illustrative rather than limiting on theinvention described herein. Scope of the invention is thus indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

Having described certain preferred and exemplary embodiments of theinvention, it will be apparent to those of ordinary skill in the artthat other embodiments incorporating the concepts disclosed herein canbe used without departing from the spirit and the scope of theinvention. The described embodiments are to be considered in allrespects only as illustrative and not limiting. Therefore, it isintended that the scope of the present invention be only limited by thefollowing claims.

1. An automatic focusing method for an optical system, comprising:performing an initial coarse focus action along a focal axis at a scanposition corresponding to a point on a surface of a slide; respectivelyperforming a plurality of subsequent fine focus actions along aplurality of focal axes at a plurality of scan positions correspondingto different points on the slide surface, wherein the performance ofeach of the fine focus actions comprises determining a fine in-focuscoordinate along the respective focal axis, determining an area of finefocus jurisdiction surrounding the respective point, and correlating thefine in-focus coordinate to the respective fine focus jurisdiction area;and determining if another scan position corresponds to a point withinat least one previously determined fine focus jurisdiction area.
 2. Themethod of claim 1, wherein each of the fine focus jurisdiction areas isgenerally elliptical in shape.
 3. The method of claim 2, wherein each ofthe fine focus jurisdiction areas has a major axis substantiallyparallel to a scan axis of the optical system and has a minor axissubstantially parallel to an index axis of the optical system.
 4. Themethod of claim 1, further comprising performing another fine focusaction along a focal axis at the other scan position if the other scanposition does not correspond to a point within at least one previouslydetermined fine focus jurisdiction area.
 5. The method of claim 4,further comprising determining another area of fine focus jurisdictionsurrounding the other point.
 6. The method of claim 1, furthercomprising determining if the other scan position corresponds to a pointwithin exactly one previously determined fine focus jurisdiction area.7. The method of claim 6, further comprising using the fine in-focuscoordinate correlated to the exactly one previously determined finefocus jurisdiction area to define another fine in-focus coordinate alonga focal axis at the other scan position if the other scan position isdetermined to correspond to a point within exactly one previouslydetermined fine focus jurisdiction area.
 8. The method of claim 7,further comprising correcting the fine in-focus coordinate correlated tothe exactly one previously determined fine focus jurisdiction area fortilt based on a global focal plane, wherein the corrected fine in-focuscoordinate is used as the other fine in-focus coordinate.
 9. The methodof claim 1, further comprising determining if the other scan positioncorresponds to a point within more than one previously determined finefocus jurisdiction area.
 10. The method of claim 9, further comprisingusing a weighted average of the fine in-focus coordinates of the morethan one previously determined fine focus jurisdiction area to defineanother fine in-focus coordinate along a focal axis at the other scanposition if the other scan position is determined to correspond to apoint within more than one previously determined fine focus jurisdictionarea.
 11. The method of claim 10, further comprising correcting theweighted average of the fine in-focus coordinates for tilt based on aglobal focal plane, wherein the corrected weighted average of the finein-focus coordinates is used as the other in-focus coordinate at theother scan position.
 12. The method of claim 1, wherein the performanceof the coarse focus action comprises determining a coarse in-focuscoordinate along the focal axis.
 13. The method of claim 12, wherein theperformance of each of the fine focus actions is based on the coarsein-focus coordinate.
 14. The method of claim 13, wherein the performanceof each of the fine focus actions comprises estimating an in-focuscoordinate along the respective focal axis as a function of the coarsein-focus coordinate.
 15. The method of claim 13, wherein the performanceof each of the fine focus actions comprises estimating an in-focuscoordinate along the respective focal axis based on a function of thecoarse in-focus coordinate and a global focal plane.
 16. The method ofclaim 1, wherein the performance of the coarse focus action comprisesrepeatedly obtaining an image of the slide at different coordinatesalong the focal axis until a coarse in-focus coordinate is determined.17. The method of claim 1, wherein the performance of each of the finefocus actions comprises estimating a fine in-focus coordinate along therespective focal axis and obtaining images of the slide at predeterminedcoordinates relative to the estimated fine in-focus coordinate along therespective focal axis.
 18. The method of claim 1, wherein theperformance of at least one of the coarse focus action and each finefocus action comprises: obtaining images of the slide at a plurality ofcoordinates along the focal axis; determining a plurality of focusscores for the respective coordinates; and selecting one of thecoordinates as an in-focus coordinate based on the focus scores.
 19. Themethod of claim 1, wherein the coordinate having a maximum focus scoreis the coordinate selected as the fine in-focus coordinate.
 20. Themethod of claim 1, wherein the slide carries a biological specimen. 21.The method of claim 1, wherein the coarse focus action and fine focusactions are performed during a single image scan.
 22. The method ofclaim 1, wherein the performance of one or both of the coarse focusaction and fine focus actions comprises moving an element of the opticalsystem relative to the slide surface to coordinates along the respectivefocal axes.
 23. The method of claim 1, wherein the performance of thefine focus actions comprises moving an element of the optical systemrelative to the slide along a scan axis to the respective scanpositions.